【作者】 王素云；

【导师】 潘崚；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 博士

【摘要】 背景:多发性骨髓瘤(MM)是发生于B细胞终末阶段浆细胞的恶性血液系统肿瘤,至今仍不可治愈。其特征之一为骨髓中多部位受恶性浆细胞累及,并浸润其他器官。临床上,约有5%的MM患者进展为浆细胞白血病。由于现有的治疗手段使MM患者总的生存时间延长,因此临床上观察到MM患者髓外浸润的发生率升高。虽然先进的治疗手段不断出现,但MM患者的预后仍然很差。众所周知,肿瘤细胞的侵袭能力不仅是恶性肿瘤的重要标志,还与较差的临床预后紧密相关。骨髓瘤细胞迁移是非常有害的病理过程,使细胞具有侵袭和播散能力。然而目前调节骨髓瘤细胞迁移至骨髓并侵袭其他部位的机制却知之甚少。如果这些机制能够被阐明,对于寻找新的治疗方法将有很大的帮助。多发性骨髓瘤患者的髓外浸润事件常常与骨髓瘤细胞增强的侵袭活性、复杂的细胞遗传学异常伴随发生。基因的异常被认为是多发性骨髓瘤疾病致病的重要因素。然而,目前使骨髓瘤细胞具有增强的侵袭活性的分子机制尚不明确。与张力蛋白同源的10号染色体缺失的磷酸酶基因(phosphatase and tensin hemology deleted on chromosome ten gene,PTEN)是近年来发现的肿瘤抑制基因,PTEN蛋白具有蛋白磷酸酶和脂质磷酸酶双重活性,其脂质磷酸酶活性可以调控细胞的增殖、凋亡以及细胞周期。其蛋白磷酸酶活性可以通过抑制丝裂原活化蛋白激酶(MAPK)、黏着斑激酶(FAK)的磷酸化等信号通路,调节细胞的迁移及黏附。PTEN可以通过多种信号传导通路影响细胞生长、分化、黏附及细胞周期进程,抑制肿瘤细胞增殖、侵袭、转移,并促进肿瘤细胞凋亡。研究发现,多种恶性肿瘤细胞株和原发肿瘤中存在PTEN基因的突变、等位基因的缺失或低表达,从而影响其肿瘤抑制功能。近年来PTEN在造血系统肿瘤中的作用逐渐受到重视。PTEN蛋白量或质的异常在造血干细胞分化、白血病发病以及白血病复发中起着重要作用。关于多发性骨髓瘤的研究中,有研究应用间期FISH,发现71例多发性骨髓瘤患者中有4例(5.6%)存在PTEN基因片段缺失,4例患者均为Durie-Salmon分期Ш期。10例原发性浆细胞白血病(PCL)患者中2例(20%)、10种人MM细胞株(HMCLs)中有两种(OCI-My6细胞和U266细胞,20%)发现有PTEN杂合性缺失。PTEN缺失被发现于进展期,并且在PCL和HMCL中发生率明显升高,这表明PTEN缺失与MM疾病的进展有关。目前,对于PTEN是否能够影响MM细胞的侵袭活性,PTEN以及PTEN信号转导通路在MM患者中的作用知之甚少。因此,本文应用腺病毒介导的PTEN基因转染人类多发性骨髓瘤细胞系RPMI8226和纯化的原代多发性骨髓瘤细胞,使MM细胞高表达PTEN基因。并且考虑到RPMI8226细胞本身表达PTEN基因,因此进一步应用PTEN-siRNA转染细胞,使PTEN基因下调,从而观察野生型PTEN对骨髓瘤细胞增殖、凋亡、细胞周期进程、侵袭活性的影响,并检测PTEN/FAK/MMP信号通路在MM细胞中的表达,以了解其可能的作用机制,为基因治疗多发性骨髓瘤提供理论依据。研究分以下三部分:第一部分PTEN、FAK在多发性骨髓瘤患者中的表达及与临床分期、髓外浸润的关系目的检测从MM患者骨髓单个核细胞经过纯化分离的骨髓瘤细胞中PTEN及FAK mRNA及其蛋白水平变化,分析MM患者中两者的关系及与多发性骨髓瘤临床分期、髓外浸润的关系,以探讨二者在多发性骨髓瘤发病中的可能作用。方法收集55例MM患者骨髓单个核细胞,应用CD138抗体将MM细胞进行纯化,同时与20个缺铁性贫血患者骨髓单个核细胞对比,并将患者按Durie-Salmon分期和是否伴有髓外浸润进行分组,并进一步进行比较。实时荧光定量PCR(FQ-PCR)法检测PTEN、FAK mRNA表达,Western blot法测定其蛋白表达水平。结果1. 55例MM患者PTEN mRNA表达水平为(0.723±0.059),对照组的表达水平为(0.984±0.359),二者相比有显著性差异(P<0.05)。FAK mRNA表达水平为(1.072±0.626),对照组的表达水平为(0.433±0.240),二者相比有显著性差异(P<0.01)。Spearman双变量相关分析结果显示,MM患者中PTEN mRNA与FAK mRNA表达水平呈显著性负相关(r=-0.560,P<0.01)。2.按Durie-Salmon分期将MM患者分为两组。Ⅰ+Ⅱ期MM患者组15例,PTEN mRNA表达水平为(0.909±0.429)。Ⅲ期MM患者组40例,PTEN mRNA表达水平为(0.653±0.347),Ⅰ+Ⅱ期MM患者组与对照组比较无统计学差异(P>0.05),Ⅲ期MM患者组与对照组比较有显著性差异(P<0.01)。Ⅰ+Ⅱ期MM患者组和Ⅲ期MM患者组比较有显著性差异(P<0.05)。Ⅰ+Ⅱ期MM患者组FAK mRNA表达水平为(0.726±0.317),Ⅲ期MM患者组为(1.224±0.667),二组分别与对照组比较差异均有统计学意义(P<0.01)。Ⅰ+Ⅱ期MM患者组和Ⅲ期MM患者组比较有显著性差异(P<0.01)。3.按是否伴有髓外浸润将所有多发性骨髓瘤患者分为两组。伴髓外浸润组共12例,PTEN mRNA表达水平为(0.656±0.296),不伴髓外浸润患者共43例,PTEN mRNA表达水平为(0.742±0.407),二组分别与对照组相比,差异均有统计学意义(P<0.05),伴髓外浸润组和无髓外浸润组相比,无统计学差异(P>0.05)。伴有髓外浸润组FAK mRNA表达水平为(1.791±0.618),无髓外浸润组为(0.878±0.468),二组分别与对照相比差异均有统计学意义(P<0.01)。伴髓外浸润组和无髓外浸润组相比差异亦具有统计学意义(P<0.01)。4. 6份对照骨髓的单个核细胞PTEN均阳性表达,平均蛋白表达水平为(0.332±0.119),T-FAK蛋白均阳性表达,平均蛋白表达水平为(0.106±0.090),p-FAK在6份对照骨髓中均未测出。5.选择12例Ⅲ期MM患者行Western blot检测,其中无髓外浸润MM患者6例,伴髓外浸润患者6例。PTEN蛋白在12例Ⅲ期MM患者中有3例不表达,9例表达,平均蛋白表达水平为(0.081±0.087),与对照组PTEN表达水平相比,差异有统计学意义(p<0.01)。T-FAK蛋白在Ⅲ期MM患者中均表达,平均蛋白表达水平为(0.257±0.119),与对照组表达水平比较差异有统计学意义(0.01<p<0.05)。p-FAK在11例患者均有不同程度的表达,平均蛋白表达水平为(0.082±0.040)。与对照组阳性率比较差异有统计学意义(x2=14.14,p<0.01)。6.无髓外浸润的6例MM患者和伴髓外浸润的6例MM患者PTEN蛋白的平均表达水平分别为(0.093±0.091)和(0.069±0.089),T-FAK蛋白的平均表达水平分别为(0.257±0.126)和(0.257±0.123),p-FAK蛋白的平均表达水平分别为(0.053±0.142)和(0.097±0.038),p-FAK蛋白的平均表达水平在两组之间有显著性差异(p<0.05), PTEN和T-FAK在两组之间无明显差异(P>0.05)。结论PTEN、FAK mRNA在多发性骨髓瘤患者中的表达水平与正常骨髓比较有显著性差异,Ⅲ期MM患者存在PTEN蛋白低表达和p-FAK蛋白高表达,MM患者PTEN和FAK表达水平可能与MM患者疾病的进展及髓外浸润密切相关,在MM的发生发展中PTEN和FAK存在互相拮抗的关系。第二部分腺病毒介导的野生型PTEN基因对多发性骨髓瘤细胞增殖、凋亡、侵袭力的影响及其机制的研究目的应用携带有野生型PTEN及绿色荧光蛋白的腺病毒(Ad- PTEN-GFP)转染RPMI8226细胞、纯化的原代多发性骨髓瘤细胞和正常人骨髓细胞,使之高表达PTEN基因,以探讨抑癌基因-野生型PTEN基因对MM细胞增殖、凋亡、细胞周期进程和侵袭活性的影响,并对其分子作用机制进行探讨。方法将Ad-PTEN-GFP或空载体腺病毒(Ad-GFP)转染RPMI8226细胞系以及纯化的原代多发性骨髓瘤细胞和正常人骨髓细胞。MTT检测细胞生长曲线;流式细胞仪检测腺病毒转染细胞的转染效率、细胞凋亡率和细胞周期分布;光镜和电镜下检测细胞形态变化;Hoechst33342荧光染色检测细胞细胞增殖及凋亡;transwell小室实验检测细胞侵袭活性;荧光定量PCR(FQ-PCR)检测mRNA水平变化;Western Blot检测蛋白水平变化。结果1.流式细胞仪检测结果证实,腺病毒转染RPMI 8226细胞在第2天达到最高转染率,MOI=100时转染效率为83.1±6.4%。转染经过纯化的原代多发性骨髓瘤细胞和正常人骨髓细胞效率较低,MOI=100时平均转染效率分别为(26.4±11.9)%和(29.3±10.5)%。2. Ad- PTEN-GFP或Ad-GFP转染RPMI8226细胞72h,PTEN mRNA表达水平在Ad-PTEN-GFP组为19.244±5.065,Ad-GFP组为1.031±0.259,二组相比存在显著性差异(p<0.01)。PTEN蛋白表达水平Ad-PTEN-GFP组为1.426±0.201, Ad-GFP组为0.783±0.176,二者相比,差异具有统计学意义(p<0.01)。3. Ad-PTEN-GFP、Ad-GFP分别转染RPMI8226细胞、纯化的原代多发性骨髓瘤细胞和正常人骨髓细胞,在MOI=100时,第4d细胞生长抑制率为42.01±7.92%,33.37±7.12%和8.42±3.09%。RPMI8226细胞和纯化的原代多发性骨髓瘤细胞与正常人骨髓细胞相比,生长抑制率存在统计学差异(P<0.01)。4. RPMI8226细胞转染Ad-PTEN-GFP、Ad-GFP 72h后,Ad-GFP组细胞呈圆形或椭圆形,体积较大,染色质疏松,透射电镜下染色质细致均匀,核仁清晰,细胞器完整,细胞微绒毛清晰可见,无凋亡的形态学改变。而转染Ad-PTEN-GFP组细胞,部分细胞体积缩小,染色质高度浓缩边集,核碎裂、溶解。透射电镜可清楚观察到细胞染色质碎裂边集,细胞器结构不完整。5. PTEN基因转染RPMI 8226细胞72 h后,各组细胞凋亡率分别为:未转染组(4.56±2.02)%,Ad-GFP组(5.51±2.43)%,Ad-PTEN-GFP组(35.02±6.80)%,Ad-PTEN-GFP组与未转染组和Ad-GFP组的细胞凋亡率相比,差异有统计学意义(P<0.01)。PTEN基因转染纯化的原代多发性骨髓瘤细胞72 h后,各组细胞凋亡率分别为:未转染组(3.62±1.99)%,Ad-GFP组(3.45±2.87)%,Ad-PTEN-GFP组(20.97±7.93)%,Ad-PTEN-GFP组与未转染组和Ad-GFP组的细胞凋亡率相比差异有统计学意义(P<0.01)。PTEN基因转染正常人骨髓细胞72 h后,各组细胞凋亡率分别为:未转染组(1.04±0.79)%,Ad-GFP组(1.32±1.01)%,Ad-PTEN-GFP组(4.01±2.48)%,Ad-PTEN-GFP组与未转染组和Ad-GFP组的细胞凋亡率相比差异有统计学意义(P<0.05)。6.以MOI=100,Ad-PTEN-GFP转染RPMI8226细胞3天,处于G2/M期的细胞比例逐渐增加,由16.20%升至51.10%,细胞周期阻滞在G2/M期。另外,细胞周期图出现亚二倍体凋亡峰,凋亡峰显示细胞凋亡率为21.6±2.35%,显著高于Ad-GFP转染组的4.8±0.74%和未转染组的4.2±0.51%(P<0.05)。7. Transwell小室实验结果显示,在MOI=100时,转染Ad-GFP与Ad-PTEN-GFP24h,RPMI 8226细胞未转染组、Ad-GFP组和Ad-PTEN-GFP组黏附于下室面的具有荧光的细胞数量分别为:50.16±7.60,52.65±7.39和23.50±6.12,未转染组和Ad-GFP组与Ad-PTEN-GFP组比较,黏附于下室面的具有荧光的细胞数量差异有明显统计学意义(P<0.01)。纯化的原代多发性骨髓瘤细胞未转染组、Ad-GFP组和Ad-PTEN-GFP组黏附于下室面的具有荧光的细胞数量分别为:57.66±11.63,56.01±8.45和30.84±7.81,未转染组和Ad-GFP组与Ad-PTEN-GFP组比较,差异有统计学意义(P<0.01)。8. mRNA表达:以MOI=100转染RPMI8226细胞后72h,结果显示在未转染组、Ad-GFP组、Ad-PTEN-GFP组,PTEN mRNA的相对表达水平分别为1.020±0.228,1.031±0.259和19.244±5.065,Ad-PTEN-GFP组与Ad-GFP组相比,PTEN mRNA表达水平显著增高(P<0.01)。凋亡相关基因Survivin mRNA相对表达水平分别为1.045±0.273,1.017±0.269和0.380±0.059 , Caspase-3 mRNA相对表达水平分别为1.018±0.227 ,0.980±0.254和2.029±0.420 , Caspase-7 mRNA相对表达水平分别为1.005±0.191,1.013±0.205和1.614±0.311。Ad-GFP组与Ad-PTEN-GFP组比较,差异有统计学意义(P<0.01,P<0.05)。FAK mRNA相对表达水平分别为0.987±0.300;0.958±0.258;0.446±0.110(P<0.05)。MMP-2 mRNA相对表达水平分别为0.992±0.251;0.984±0.270;0.469±0.147(P<0.05)。MMP-9 mRNA相对表达水平分别为0.983±0.238;0.972±0.257;0.366±0.104(P<0.01)。9.蛋白表达:以MOI=100转染RPMI8226细胞后72h,在未转染组、Ad-GFP组、Ad-PTEN-GFP组,PTEN相对表达水平分别为0.732±0.159;0.783±0.176;1.426±0.201,Ad-PTEN-GFP组与Ad-GFP组相比,PTEN蛋白表达水平显著增高(P<0.01)。Survivin蛋白表达水平分别为0.348±0.081,0.356±0.090和0.082±0.023 ( P<0.01 )。T-FAK蛋白表达水平分别为1.178±0.302,1.137±0.282和0.730±0.197(P<0.05)。p-FAK蛋白表达水平分别为0.289±0.083,0.304±0.095和0.068±0.027(P<0.01)。MMP-2蛋白表达水平分别为0.622±0.220,0.691±0.238和0.155±0.076(P<0.01)。MMP-9蛋白表达水平分别为0.358±0.094,0.339±0.143和0.098±0.060(P<0.05)。10. Caspase-3、-7蛋白活性:Ad-PTEN-GFP以MOI=100转染RPMI 8226细胞72h,Caspase-3/7活性为(0.786±0.081),高于Ad-GFP组(0.373±0.039)和未转染组(0.370±0.034),差异有统计学意义(P<0.01)。结论过表达PTEN基因能够明显抑制RPMI8226细胞和原代多发性骨髓瘤细胞增殖,诱导其凋亡,并调控多种凋亡相关分子,使细胞周期阻滞在G2/M期,细胞侵袭能力降低,其分子机制可能与PTEN抑制FAK/MMP信号途径有关。第三部分抑制PTEN基因表达对多发性骨髓瘤细胞RPMI8226增殖、侵袭力的影响及其机制的研究目的应用PTEN-siRNA使PTEN基因表达下降,探讨PTEN基因对RPMI8226细胞增殖、细胞周期和侵袭力的影响,并对其分子作用机制进行探讨。方法应用针对小鼠PTEN序列的特异性小干扰RNA(small interfering RNA,siRNA)转染RPMI 8226细胞,阻断内源性PTEN的表达。MTT检测细胞生长曲线;流式细胞仪检测细胞周期分布;transwell小室实验检测细胞侵袭活性;荧光定量PCR(FQ-PCR)检测mRNA水平变化,Western Blot检测蛋白水平变化。结果1.检测PTEN mRNA及蛋白检测来测定PTEN-siRNA的转染效果。结果显示在PTEN-siRNA转染RPMI8226细胞48h,PTEN mRNA表达水平在NS-siRNA转染组为1.107±0.306 , PTEN-siRNA转染组为0.143±0.045,二组相比存在显著性差异(p<0.01)。PTEN蛋白表达水平在NS-siRNA转染组为0.699±0.130,PTEN-siRNA转染组为0.089±0.025,二者相比差异具有统计学意义(p<0.01)。2. MTT结果显示,转染PTEN-siRNA第4d,细胞生存率为(141.55±8.34)%,NS-siRNA转染组和PTEN-siRNA转染组OD 490值比较有统计学意义(P<0.01)。3.细胞周期分析显示PTEN-siRNA转染RPMI8226细胞48h后,与未转染细胞相比,处于G0/G1期细胞比例升高,G2/M期和S期细胞比例降低,凋亡峰显示凋亡的细胞比例减少。4. PTEN-siRNA转染RPMI8226细胞后24h,未转染组、NS-siRNA转染组和PTEN-siRNA转染组黏附于下室面的细胞数量分别为:49.33±7.63、47.17±7.76和79.50±11.89,NS-siRNA和PTEN-siRNA二组相比,差异有显著统计学意义(P<0.01)。5. mRNA表达:PTEN-siRNA转染RPMI8226细胞后48h,未转染组、NS-siRNA组、PTEN-siRNA组,PTEN mRNA相对表达水平分别为1.049±0.248;1.107±0.306;0.143±0.045,NS-siRNA组和PTEN-siRNA组相比有统计学意义(P<0.01)。凋亡相关基因Survivin mRNA相对表达水平分别为1.013±0.207,1.008±0.191和2.534±0.438,Caspase-3 mRNA相对表达水平分别为1.008±0.198,0.979±0.174和0.473±0.089,Caspase-7 mRNA相对表达水平分别为1.015±0.196,1.003±0.204和0.510±0.090。NS-siRNA和PTEN-siRNA二组相比,差异有显著统计学意义(P<0.01) FAK mRNA相对表达水平分别为0.979±0.308;0.940±0.301;2.176±0.61(2P<0.01)。MMP-2 mRNA相对表达水平分别为1.002±0.213;0.998±0.206;2.793±0.74(0P<0.01)。MMP-9 mRNA相对表达水平分别为1.086±0.219;1.029±0.280;1.980±0.733(P<0.05)。6.蛋白表达:PTEN-siRNA转染RPMI8226细胞后48h,未转染组、NS-siRNA组、PTEN-siRNA组,PTEN蛋白相对表达水平分别为0.647±0.124,0.699±0.130和0.089±0.025,NS-siRNA组与PTEN-siRNA组相比有显著性差异(P<0.01)。Survivin蛋白表达水平分别0.312±0.089,0.324±0.086和1.247±0.321(P<0.01)。T-FAK蛋白表达水平分别为0.983±0.217;0.945±0.226;1.637±0.380(P<0.05)。p-FAK蛋白表达水平分别为0.345±0.086;0.390±0.091;1.054±0.272(P<0.01)。MMP-2蛋白表达水平分别为0.233±0.080;0.295±0.092;1.265±0.383(P<0.01)。MMP-9蛋白表达水平分别为0.237±0.083;0.221±0.084;0.544±0.161(P<0.05)。7. Caspase-3、-7蛋白活性:PTEN-siRNA转染RPMI 8226细胞72h,Caspase-3/7活性为(0.073±0.008),低于NS-siRNA组(0.369±0.038)和未转染组(0.370±0.034),差异有统计学意义(P<0.01)。结论转染PTEN干扰RNA能够促进RPMI8226细胞增殖,并使细胞凋亡减少,细胞周期检测结果提示处于G0/G1期细胞比例升高,G2/M和S期细胞减少,细胞侵袭能力增加。可能与PTEN表达水平降低,并进一步引起凋亡相关基因分子以及FAK/MMP信号转导通路信号分子mRNA和蛋白表达水平的异常有关。更多还原

【Abstract】 Background: Multiple myeloma is a kind of incurable malignancy of end stage B-lineage cells, and characterized by an accumulation of neoplastic plasma cells in the bone marrow and invasion into other organs. Plasma leukemia develops in about 5% of MM patients as a terminal disease manifestation. Other extramedullary infiltrations are observed with increasing frequency as the overall survival of MM patients has been prolonged by current therapy. Prognosis of patients with MM remains poor despite the advanced therapeutic protocols. Invasiveness of tumor cells is not only an important indicator of the malignancy, but is also closely related with poor clinical prognosis. Migration of myeloma cells is a very harmful disease process fundamental to the invasion and dissemination of myeloma cells. However at present, little is known about the mechanisms regulating myeloma cell migration in the bone marrow and metastasizing to secondary sites. If these mechanisms are elucidated, it would be of great significance and very beneficial for looking for new treatment strategies. Extramedullary disease is almost always accompanied with complex cytogenetic abnormalities. Genetic abnormality is considered to be one of the important pathogenic factors for the genesis of MM. However, the identities of molecular alterations that allow these cancer cells to have increased metastatic potential are not clear.Phosphatase and tensin hemology deleted on chromosome ten gene (PTEN) is a recently identified tumor suppressor gene. PTEN protein has protein phosphatase and lipid phosphatase dual activity. PTEN can inhibit proliferation, induce apoptosis and regulate cell cycle progression by lipid phosphatase activity. The protein phosphatase activity of PTEN can regulate cellular bioactivities such as migration and focal adhesion, by targetting mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK) pathway and inhibiting phosphorylation of several proteins. Briefly PTEN can regulate the growth, differentiation, adhesion and cell cycle of normal cells; supress the proliferation, invasion and metastasis of tumor cells; induce apoptosis of tumor cells.Previous studies found that genetic mutation/deletion or lower expression of PTEN gene is so common in several types of human solid cancers, indicating that PTEN is one of the most frequently candidate of tumor suppressors. The function of PTEN gene in the genesis of hematopoietic malignancies such as leukemia has been focused generally. PTEN protein played an important role in the differentiation of hematopoietic stem cells and in the prevention of leukemia genesis and relapse. In MM, previous studies found hemizygous deletions of PTEN in 5% of MM patients, in 20% of plasma cell leukemias (PCL) and in 20% of human myeloma cell lines (HMCLs) as determined by inter-phase cIg-FISH methods. PTEN deletion was detected in advanced disease, such as PCL or HMCLs, suggesting that PTEN alterations occurred as a secondary change associated with disease progression in MM.However, little is known about the relationship between the genesis of MM and the activity of PTEN signal transduction pathways and whether PTEN affect the invasion ability of MM cells. To explore these questions, RPMI 8226 cells (a human MM cell line) and purified myeloma cells from MM patients were transfected with a recombinant adenovirus-PTEN vectors containing green fluorescent protein (Ad-PTEN-GFP) or an adenovirus vectors only expressing green fluorescent protein (Ad-GFP). Since RPMI 8226 cells natively express the PTEN gene, we down regulated the expression of wild type PTEN by using PTEN-siRNA as a control. The effects and the mechanism of wild-type PTEN gene on the proliferation, apoptosis, cell cycle progression and invasion activity of MM cells were studied. The result of this study may provid an important theoretical basis for MM therapy with PTEN gene. This paper consists of the following three parts:Part one: Expression levels of PTEN, FAK in multiple myeloma patients and its relationship with clinical stage and extramedullary infiltrationObjective To investigate PTEN, FAK mRNA and protein levels in MM patients, analyze their relationship with clinical stage of multiple myeloma and extramedullary infiltration, and explore its role in Multiple Myeloma.Methods Bone Marrow Mononuclear Cells (BMMNCs) were isolated from 55 patients with MM and 20 Control patients. Bone marrow CD138+ plasma cells were purified from the isolated BMMNCs with an immuno-magnetic method using anti-CD138 monoclonal antibody (mAb)–coated microbeads. MM patients were grouped according to Durie-Salmon stage, with or without extramedullary infiltration. The mRNA expression levels of PTEN, FAK were measured by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR). The expression levels of PTEN, T-FAK and p-FAK were determined by western blotting method.Results1. The expression level of PTEN mRNA was (0.723±0.059) in 55 patients of MM and (0.984±0.359) in the control group. The expression level of PTEN mRNA was significantly lower in MM patients than that in control group (P<0.05). The expression level of FAK mRNA was (1.072±0.626) in MM patients and (0.433±0.240) in control group. There was a statistically significant difference in the expression level between these two groups (P<0.01). Spearman bivariate correlation analysis showed that the expression levels of PTEN and FAK were significantly negatively correlated in MM patients (r =- 0.560, P <0.01).2. All MM patients were divided into two groups according to Durie-Salmon stage. The expression level of PTEN mRNA was (0.909±0.429) in MM patients of stageⅠ+Ⅱ(15 cases), and (0.653±0.347) in MM patients ofⅢphase (40 cases). When the expression level of PTEN mRNA in MM patients of stageⅠ+Ⅱcompared with control group, the difference was not statistically significant (P>0.05). When the expression level of PTEN mRNA in stageⅢof MM patients compared with control group, the differenc was statistically significant (P<0.01). The difference of PTEN mRNA expression level between stageⅠ+Ⅱof MM patients and stageⅢof MM patients was statistically significant(P<0.05).The expression level of FAK mRNA was (0.726±0.317) in MM patients of stageⅠ+Ⅱand (1.224±0.667) in MM patients of stageⅢ. Compared with the control group, the differenc was statistically significant (P <0.01). Furthermore, the expression level of FAK mRNA was statistically significant difference when MM patients of stageⅠ+Ⅱcompared with MM patients of stageⅢ(P<0.01).3. The MM patients were divided into two groups according to whether having extramedullary infiltration or not. The expression level of PTEN mRNA was (0.656±0.296) in 12 MM patients with extramedullary infiltration. Compared with the control, the differenc was statistically significant (P<0.05). The expression levels of PTEN mRNA were (0.742±0.407) in 43 MM patients without extramedullary infiltration. Compared with the control, the differenc was statistically significant (P<0.05). When MM patients with extramedullary infiltration compared with MM patients without extramedullary infiltration, the difference between these two group was not statistically significant (P> 0.05).FAK mRNA expression level was (1.791±0.618) in MM patients with extramedullary infiltration, and (0.878±0.468) in MM patients without extramedullary infiltration, and the difference between these two group was statistically significant (P<0.01). And the expression level of FAK mRNA in each patients group was significantly higher than that in control group (P <0.01).4. PTEN and T-FAK protein expressed in the bone marrows of 6 selected normal controls, and the average expression level of PTEN protein was (0.332±0.119) and T-FAK protein was (0.106±0.090) respectively. The expression of p-FAK protein was not detected. 5. PTEN- and FAK- protein expression level was measured in 12 MM patients of stageⅢby Western blot. Amoung these 12 patients, 6 had no extramedullary infiltration while the other 6 had. PTEN protein expression was detected in 9 patients and the average protein expression level was (0.081±0.087), which was statistically lower than that in control group (P<0.01). T-FAK protein were expressed in all thses 12 patients, and the average protein expression level was (0.257±0.119), which was statistically significant higher than that in control group (0.01<P<0.05). The p-FAK protein expressed in 11 patients, and the average protein level was (0.082±0.040), when compared with the control group, the differenc was statistically significant (x2 = 14.14, P <0.01).6. In the 6 patients without extramedullary infiltration and the 6 cases with extramedullary, the average PTEN protein level was (0.093±0.091) and (0.069±0.089), respectively. T-FAK protein level was (0.257±0.126) and (0.257±0.123), and p-FAK protein level was (0.053±0.142) and (0.097±0.038) respectively. A statistically significant difference was found in the expression level of p-FAK protein between the two groups (P< 0.05), but not in PTEN and T-FAK protein expression level (P>0.05).Conclusions There was statistically significant difference of PTEN and FAK mRNA expression between multiple myeloma patients and normal controls. The expression level of PTEN protein was lower and p-FAK protein was higher in MM patients of stageⅢthan that of in controls. The abnormal expression level of PTEN and FAK may be associated with the progression of the disease and extramedullary infiltration in MM patients. PTEN and FAK may be mutually antagonistic in the occurrence and development of MM.Part two: The effect of wild-type PTEN gene transfection on proliferation, apoptosis, invasion ability of multiple myeloma cells and its mechanismObjective RPMI 8226 cells and purified myeloma cells from MM patients were transfected with a recombinant adenovirus-PTEN vectors containing green fluorescent protein (Ad-PTEN-GFP) or an adenovirus vectors only expressing green fluorescent protein (Ad-GFP), explore the effects of wild-type PTEN gene on proliferation, apoptosis, cell cycle and invasion activity of RPMI 8226 cells and purified myeloma cells from MM patients and the possible molecular mechanism.Methods Ad-PTEN-GFP or Ad-GFP was transfected into RPMI 8226 cells, purified myeloma cells from MM patients and the bone marrow mononeuclear cell of healthy controls. The growth inhibition rate was measured by MTT assay after the cells were transfected with PTEN gene. The transfection efficiency, apoptosis rate and cell cycle distribution were assessed by flow cytometry (FCM). Cell morphology was detected by light microscope and electron microscope. Cell proliferation and apoptosis were detected by Hoechst33342 staining. Transwell chamber test was used to meacure MM cell invasion activity. The mRNA expression levels of PTEN, FAK, MMP-2 and MMP-9 were detected by FQ-PCR. The protein expression levels of PTEN, FAK, p-FAK, MMP-2 and MMP-9 were detected by western blot.Results1. At the condition of multiple of infection (MOI)=100, at the second days of transfection, the transfection efficiency with adenovirus reached the top level: (83.1±6.4)% for RPMI 8226 cells, (26.4±11.90% for purified fresh myeloma cells and (29.3±10.5)% for the BMMNC of controls.2. After RPMI 8226 cells had been transfected with Ad-GFP or Ad-PTEN-GFP for 72 h, the expression level of PTEN mRNA and protein in RPMI 8226 cells from each group was measured by FQ-PCR and western blot. PTEN mRNA level was 19.244±5.065 vs 1.031±0.259 (p<0.01), and protein level was 1.426±0.201 vs 0.783±0.176 (p<0.01) respectively in Ad-PTEN-GFP transfected RPMI 8226 cells vs Ad-GFP transfected RPMI 8226 cells.3. Ad-PTEN-GFP, Ad-GFP were transfected into RPMI8226 cells, purified myeloma cells from MM patients and BMMNCs from healthy control, maximal growth inhibition rate was (42.01±7.92) % and (33.37±7.12) % respectively in Ad-PTEN-GFP transfected RPMI 8226 cells and the Ad-PTEN-GFP transfected purified myeloma cells from MM patients at the 4th day. Maximal growth inhibition was (8.42±3.09) % in Ad-PTEN-GFP transfected BMMNCs from healthy control at the 4th day, which was statistically significant lower compared with that of RPMI 8226 cells and purified myeloma cells from MM patients (p<0.01).4. After transfected with Ad-PTEN-GFP for 3d, the untransfected cells and Ad-GFP transfected MM cells displayed excellent growth state. The non-apoptotic cells were rounded and light blue. The chromatin was delicate and homogeneous, the nucleolus was clear, the organelles complete and the microvilli on the cells were clear and visible under transmission electron microscope. Ad-PTEN-GFP transfection induced typical apoptosis of MM cells, the apoptotic cells were bright blue and exhibited cell shrinkage and detachment, with lobular nuclear, debris-like, nuclear margination, nuclear condensation and fragmentation. Under the transmission electron microscope, it was shown that the microvilli on the cells disappeared and structure of the organelles was incomplete.5. After transfected with Ad-PTEN-GFP for 72 h, the apoptosis rate of RPMI8226 cells was (4.56±2.02)% in untransfected group, (5.51±2.43)% in Ad-GFP group, (35.02±6.80)% in Ad-PTEN-GFP group, and the difference of apoptosis rate between Ad-PTEN-GFP group and Ad-GFP group was significantly different (P<0.01). Accordingly, after the transfection for 72 hours, the apoptosis rate of the purified primary myeloma cells was (3.62±1.99) % in untransfected group, (3.45±2.87)% in Ad-GFP group and (20.97±7.93)% in Ad- PTEN-GFP group, and the diference of apoptosis rate between Ad-PTEN-GFP group and Ad-GFP group was significantly different (P<0.01). Samely, after the transfection for 72 hours, the apoptosis rate of the BMMNCs of control was (1.32±1.01) % in untransfected group, (1.04±0.79) % in Ad-GFP group and (4.01±2.48) % in Ad-PTEN-GFP group. And the diference of apoptosis rate between RPMI 8226 cells, or BMMNCs of control, and between the purified primary myeloma cells and BMMNCs of control was significantly different (P<0.05). 6. After RPMI8226 cells had been transfected with Ad-PTEN-GFP for 72 h, cell cycle distribution showed the percentage of cells in G2/M phase increased from 16.20% to 51.10%. Cell cycle of Ad-PTEN-GFP transfected RPMI8226 cells was arrest in G2/M phase and there was an apoptotic sub G1 peak in cell cycle diagram. Cell cycle diagram showed that the apoptosis rate was (21.6±2.35) % in Ad-PTEN-GFP, which was significantly higher than the (4.8±0.74)% in Ad-GFP group and the (4.2±0.51) % in untransfected group (P<0.05).7. For the transwell chamber test, after the cells had been transfected with Ad-PTEN-GFP for 24 h, the average number of fluorescent RPMI8226 cells which migrated through the matrigel and filter from the upper chamber to the lower chamber in the untransfected control group, the Ad-GFP group and the Ad-PTEN-GFP group was 50.16±7.60, 52.65±7.39 and 23.50±6.12 respectively. The difference between the Ad-GFP group and the Ad-PTEN-GFP group was statistically significant (P<0.01). Samely, The average number of fluorescent purified myeloma cells from MM patients which migrated through the matrigel and filter from the upper chamber to the lower chamber in the untransfected control group, the Ad-GFP group and the Ad-PTEN-GFP group was 57.66±11.63, 56.01±8.45 and 30.84±7.81 respectively. And the difference between the Ad-GFP group and the Ad-PTEN-GFP group was statistically significant (P<0.01).8. The expression level of target mRNAs: After RPMI8226 cells had been transfected with Ad-PTEN-GFP for 72 h, the expression level of PTEN mRNA was 1.020±0.228, 1.031±0.259, 19.244±5.065 in untransfected group, Ad-GFP group and Ad-PTEN-GFP group respectively.In untransfected group, Ad-GFP group and Ad-PTEN-GFP group, the expression level of Survivin mRNA was 1.045±0.273, 1.017±0.269 and 0.380±0.059 respectively. Caspase-3 mRNA level was 1.018±0.227, 0.980±0.254 and 2.029±0.420 respectively. Caspase-7 mRNA level was 1.005±0.191, 1.013±0.205 and 1.614±0.311 respectively. The expression level of FAK mRNA was 0.987±0.300, 0.958±0.258 and 0.446±0.110 respectively. The expression level of MMP-2 mRNA was 0.992±0.251, 0.984±0.270 and 0.469±0.147 respectively. The expression level of MMP-9 mRNA relative expression level was 0.983±0.238, 0.972±0.257 and 0.366±0.104 respectively. The difference was statistically significant between the Ad-PTEN-GFP group and Ad-GFP group (P<0.01, P<0.05).9. The expression level of target proteins: After RPMI8226 cells had been transfected with Ad-PTEN-GFP for 72 h, the expression level of PTEN protein was 0.732±0.159, 0.783±0.176 and 1.426±0.201 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. Survivin protein level was 0.348±0.081, 0.356±0.090 and 0.082±0.023 in untransfected group, Ad-GFP group and Ad-PTEN-GFP group (P<0.01).T-FAK protein level was 1.178±0.302, 1.137±0.282 and 0.730±0.197 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. The expression level of p-FAK protein was 0.289±0.083, 0.304±0.095 and 0.068±0.027 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. MMP-2 protein level was 0.622±0.220, 0.691±0.238 and 0.155±0.076 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. MMP-9 protein level was 0.358±0.094, 0.339±0.143 and 0.098±0.060 respectively in the untransfected group, Ad-GFP group and Ad-PTEN-GFP group. The difference was statistically significant between the Ad-PTEN-GFP group and Ad-GFP group (P<0.01, P<0.05).10. After transfection with Ad-PTEN-GFP for 72 h, the activitay of caspase-3/7 (0.786±0.081) in Ad-PTEN-GFP transfected RPMI 82226 cells was increased significantly compared with that of in the Ad-GFP transfected cells (0.373±0.039) and the Untransfected cells(0.370±0.034)(P<0.01).Conclusions Over expression PTEN gene in RPMI8226 cells and purified myeloma cells from MM patients could inhibit the proliferation, induce apoptosis. With the transfection of wild type PTEN gene, cell cycle of the transfected cells was arrested in G2/M phase, apoptosis occurred, the invasion ability decreased, the expression levels of many apoptosis related genes were changed, which might be related to the inhibition of FAK / MMP signal pathway by the high expression of PTEN.Part three: Effect of Silencing PTEN gene expression on the ability of proliferation, invasion of RPMI8226 cells and its mechanismObjective RPMI 8226 cells were transfected with mouse PTEN specific siRNA (PTEN-siRNA) to down regulated the expression of wild type PTEN, to investigate the effect of PTEN gene on the proliferation, cell cycle and invasion activity of RPMI8226 cells, and explore its molecular mechanism.Methods RPMI 8226 cells were transfected with PTEN-siRNA or non-specific siRNA (NS-siRNA) to knockdown the expression of wild type PTEN. Cell growth curve was measured by MTT assay. Cell cycle distribution was assessed by flow cytometry (FCM). Transwell chamber test was used to meacure MM cell invasion activity. The mRNA expression levels were detected by FQ-PCR. The protein expression levels were detected by western blots.Results1. Detection of PTEN mRNA and protein to evaluate the efficency of PTEN-siRNA transfection. After RPMI 8226 cells had been transfected with PTEN-siRNAs or NS-siRNAs for 48 h, the expression level of PTEN mRNA and protein in RPMI 8226 cells from each group was measured by FQ-PCR and western blot. PTEN mRNA level was 1.107±0.306 vs 0.143±0.045 (p<0.01), and protein level was 0.699±0.130 vs 0.089±0.025 (p<0.01) respectively in NS-siRNAs transfected RPMI 8226 cells vs PTEN-siRNAs transfected RPMI 8226 cells.2. RPMI8226 cells were divided into three group: untransfected group, NS-siRNA transfected group and PTEN-siRNA transfected group. After RPMI 8226 cells had been transfected with PTEN-siRNAs for 4 days, cell survival rate in PTEN-siRNA transfected group was (141.55±8.34)%. The OD 490 values were significant difference between the NS-siRNA transfected group and the PTEN-siRNA transfected group (P<0.01).3. After RPMI8226 cells had been transfected with PTEN-siRNA for 48 h, cell cycle analysis showed that the percentage of RPMI8226 cells in G0/G1 phase was increased and the percentage of RPMI8226 cells in G2/M phase and S phase was decreased. Sub-diploid apoptotic peak showed that the percentage of apoptotic cells was decresed.4. For transwell chamber test, after RPMI8226 cells had been transfected with PTEN-siRNA for 24 h, the average number of RPMI8226 cells which migrated through the matrigel and filter from the upper chamber to the lower chamber in the untransfected control group, the NS-siRNA group and the PTEN-siRNA group was 49.33±7.63, 47.17±7.76 and 79.50±11.89 respectively. The difference in the cell number between NS-siRNA group and the PTEN-siRNA group were statistically significant (P<0.01).5. The expression level of target mRNA: After RPMI8226 cells had been transfected with PTEN-siRNA for 48h, the expression level of PTEN mRNA was 1.049±0.248, 1.107±0.306 and 0.143±0.045 respectively in the untransfected group, NS-siRNA group, and PTEN-siRNA group.In untransfected group, NS-siRNA group, and PTEN-siRNA group, the expression level of Survivin mRNA was 1.013±0.207, 1.008±0.191 and 2.534±0.438 respectively. Caspase-3 mRNA level was 1.008±0.198, 0.979±0.174 and 0.473±0.089 respectively. Caspase-7 mRNA level was 1.015±0.196, 1.003±0.204 and 0.510±0.090 respectively. Accordingly, in untransfected group, NS-siRNA group, and PTEN-siRNA group, the expression level of FAK mRNA was 0.979±0.308, 0.940±0.301 and 2.176±0.612 respectively. The expression level of MMP-2 mRNA was 1.002±0.213, 0.998±0.206 and 2.793±0.740 respectively in the untransfected group, NS-siRNA group, and PTEN-siRNA group. The expression level of MMP-9 mRNA was 1.086±0.219, 1.029±0.280 and 1.980±0.733 respectively in the untransfected group, NS-siRNA group, and PTEN-siRNA group. The difference was statistically significant between the PTEN-siRNA group and NS-siRNA group (P<0.01, P<0.05).6. The expression level of target protein: After RPMI8226 cells had been transfected with PTEN-siRNA for 48h, the expression level of PTEN protein was 0.647±0.124, 0.699±0.130 and 0.089±0.025 respectively in the untransfected group, NS-siRNA group and PTEN-siRNA group. Survivin protein level was 0.312±0.089, 0.324±0.086 and 1.247±0.321 (P<0.01). T-FAK protein level was 0.983±0.217, 0.945±0.226 and 1.637±0.380 respectively. The expression level of p-FAK protein was 0.345±0.086, 0.390±0.091 and 1.054±0.272 respectively. MMP-2 protein level was 0.233±0.080, 0.295±0.092 and 1.265±0.383 respectively. MMP-9 protein level was 0.237±0.083, 0.221±0.084 and 0.544±0.161 respectively. The difference was statistically significant between the PTEN-siRNA group and NS-siRNA group (P<0.01, P<0.05).7. After transfection with PTEN-siRNA for 72 h, the activitay of caspase-3/7 (0.073±0.008) in PTEN-siRNA transfected RPMI 82226 cells was decreased significantly compared with that of the NS-siRNA transfected cells (0.369±0.038) and the Untransfected cells(0.370±0.034)(P<0.01).Conclusions Down regulated the expression of wild type PTEN by using PTEN-siRNA can significantly enhance the proliferation of RPMI8226 cells, decrease cell apoptosis, increase the percentage of cells in G0/G1 phase and decrease the percentage of cells in G2/M and S phase, enhance the cell invasion ability, indicate that the deceased expression of wild type PTEN might further regulate apoptosis related genes, and increase the expression level of FAK, MMP-2 and MMP-9 mRNA and protein, and stimulate the proliferation and invasion of myeloma cells.更多还原