【作者】 张克勤；

【导师】 徐祗顺；

【作者基本信息】 山东大学 ， 外科学， 2009， 博士

【摘要】 目的探讨肿瘤微环境对骨髓间充质干细胞(MSCs)的趋化性以及MSCs在肿瘤微环境中分化为肌纤维母细胞的体外研究。方法于雄性新西兰大白兔股骨大转子处抽取骨髓约1ml,采用密度梯度离心法联合贴壁培养法分离扩增MSCs,用流式细胞仪检测培养的MSCs的表面抗原。无菌方法摘取VX2瘤兔的新鲜肿瘤组织进行原代培养扩增,并收集其培养上清。采用Transwell法比较对照组、体积分数为30%和50%的VX2上清培养基对F2代MSCs的趋化性;根据Transwell结果,将F2代MSCs用体积分数为30%VX2上清培养基刺激,分别在7天、14天时采用RT-PCR和Westernblot方法检测肌纤维母细胞标记物α-SMA和Vimentin的表达情况。结果MSCs细胞呈梭形,培养的F2代MSCs流式鉴定结果为CD29(+),CD44(+),CD45(-),CD106(+);VX2细胞呈多边形或长梭形;Transwell试验中发现:镜下体积分数为30%VX2上清组穿过基质胶的细胞数明显多于对照组和50%组,这一结果被随后进行的比色测定结果进一步证实;RT-PCR和Westernblot结果均发现:用30%VX2上清培养基刺激7天后,MSCs表达α-SMA和Vimentin的水平均明显强于对照组(P＜0.05);而刺激14天后,MSCs表达α-SMA和Vimentin的水平均进一步增强(P＜0.05)。结论肿瘤微环境对MSCs有一定的趋化性,MSCs可以在肿瘤微环境的刺激下分化为肌纤维母细胞。意义本实验在一定程度上表明:在肿瘤微环境的诱导下,MSCs能够进入肿瘤组织并在其刺激下转化为肌纤维母细胞,这可能是MSCs促进肿瘤发生发展的一个途径;MSCs可能是肿瘤/癌活化肌纤维母细胞的一个重要来源。目的探讨骨髓间充质干细胞(MSCs)在肿瘤发生发展过程中的作用及其是否可以转化为血管内皮细胞。方法随机将20只雄性新西兰大白兔分为2组:实验组和对照组。每只动物抽取骨髓培养MSCs后,采用VX2瘤块包埋法建立膀胱肿瘤模型。模型建立1周后,实验组回输DAPI标记的自身F2代MSCs,而对照组回输培养液。在模型建立2、4周后分别用经腹超声方法检测肿瘤大小。4周后处死所有动物,采用双重免疫荧光方法检测实验组中植入的MSCs能否转化为血管内皮细胞。另外,将实验组和对照组膀胱肿瘤标本做成石蜡切片,每个动物随机抽取3张,HE染色后,计数高倍视野下(×400)血管数目,比较两组动物肿瘤组织血管密度的差异。体外实验中原代培养MSCs和VX2细胞,收集新鲜VX2上清,实验组是用体积分数为10%胎牛血清、30%VX2上清的低糖DMEM培养的F2代MSCs,对照组是用体积分数为10%胎牛血清的低糖DMEM培养的F2代MSCs,分别在7天、14天时采用Westernblot方法比较两组细胞血管内皮细胞标记物CD146的蛋白表达情况。结果实验组和对照组在2周时肿瘤最大径分别是0.77±0.1 5cm和0.71±0.15cm,两组间没有明显统计学差异(P＞0.05)。4周时实验组肿瘤最大径(3.82±0.94cm)明显大于对照组(2.28±0.54cm)(P＜0.05)。实验组冰冻切片显示DAPI/CD146双重免疫荧光阳性的细胞存在于血管腔表面,表明植入的MSCs已转化为血管内皮细胞。实验组和对照组的肿瘤组织血管密度分别是10.1±0.70/0.2mm~2和8.24±0.81/0.2mm~2,两组间存在明显统计学差异(P＜0.05);体外实验中Westernblot结果发现:实验组的MSCs用体积分数30%VX2上清培养基刺激7天后,CD146的蛋白表达水平明显强于对照组(P＜0.05);而刺激14天后,CD146蛋白表达水平进一步增强(P＜0.05)。结论MSCs能够在肿瘤微环境中分化为血管内皮细胞,促进血管生成,这可能是其促进肿瘤发生发展的主要途径。目的探讨良性前列腺增生(BPH)患者膀胱内前列腺突入(IPP)程度测定对膀胱出口梗阻及膀胱功能的预测与评价。方法分析206例初次就诊的良性前列腺增生患者的资料,根据经腹超声测量的IPP值将患者分为两组:明显突入组(IPP＞1cm)和不明显突入组(IPP≤1cm),分析两组间临床资料及尿动力学检查结果间的关系。结果临床资料显示:明显突入组(sIPP)和不明显突入组(nsIPP)患者在前列腺体积(73.7±35.9cm~3 vs 62.8±36.5 cm~3)、前列腺特异性抗原(PSA)(1.81±0.67ng/mlvs 1.64±0.36 ng/ml)、剩余尿量(290.2±217.2ml vs 228.2±167.9ml)、急性尿潴留(33.3%vs 18.0%)及膀胱小梁化(23.1%vs 11.7%)五个方面的差异均有明显统计学意义(P＜0.05)。尿动力学检查结果显示:两组患者的排尿期最大尿流率(Qmax)(7.6±4.1ml/s vs 9.1±3.6ml/s)、膀胱过度活动症发生率(82.1%vs 17.2%)、膀胱顺应性降低率(35.9%vs 12.5%)、最大逼尿肌压力(Pdet.max)(109.8±84.9cmH2O vs 84.9±44.1 cmH2O)及膀胱出口梗阻指数(BOOI)(75.2±27.1 vs 65.9±34.6)间的差异均有统计学意义(P＜0.05)。结论IPP可以作为初步预测和评价膀胱出口梗阻程度及膀胱功能的一项指标。前列腺明显突入膀胱的BPH患者膀胱出口梗阻及膀胱功能受损的程度明显高于无明显突入患者,对于前列腺明显突入膀胱尤其是合并急性尿潴留的BPH患者应及早采取外科手术治疗。更多还原

【Abstract】 OBJECTIVE To investigate the tropism of mesenchymal stem cells(MSCs) to tumor microenvironment and the feasibility of bone marrow mesenchymal stem cells differentiating into myofibroblast in vitro.METHODS 1ml bone marrow was taken from greater trochanter of male New Zealand rabbit and MSCs were obtained by density gradient centrifugation and cultured routinely;the surface markers were tested by flow cytometry.VX2 tumor was aseptically excised and primary cultured.The tropism of MSCs for control group, 30%and 50%VX2 conditioned medium were determined by using Transwell migration assay.MSCs were incubated in 30%VX2 conditioned medium for 7 or 14 days.The mRNA levels and protein expression ofα-SMA and Vimentin were measured by RT-PCR and Westernblot methods.RESULTS MSCs presented a spindle shape.The cultured MSCs were CD29(+), CD44(+),CD45(-),CD106(+).VX2 cells showed spindle or polygon shape.In the Transwell test,we found that the migrated cells appeared more in 30%VX2 conditioned medium group than other groups under microscope,which was further confirmed by the results of colorimetric assay.The mRNA levels and protein expression ofα-SMA and Vimentin both significantly increased in the 7 days group than the control group(P＜0.05),which further increasd in the 14 days group(P＜0.05).CONCLUSION MSCs show tropism for tumor microenvironment and can differentiate into myofibroblast in tumor microenvironment in vitro.SIGNIFICANCE This experiment suggests that MSCs could migrate to tumor and then differentiate into myofibroblast under tumor microenvironment,which might be a pathway of MSCs promoting the growth of tumor.It also suggestes that MSCs may be the precursors of the tumor/carcinoma associated myofibroblasts. OBJECTIVE To investigate the effect of mesenchymal stem cells(MSCs) in the process of tumor development and the possibility of MSCs differentiating into vascular endothelial cells in tumor microenvironment.METHODS 20 male New Zealand rabbits were randomly divided into two groups: test group and control group.MSCs were isolated and cultured by bone marrow cell adherence.The bladder tumor models were built by embedding vx2 mass in swelled bladder mucosa in all rabbits.One week later,4’,6-diamidino-2-phenylindole labeling MSCs were transplanted into tumor tissue in test group(n=10).Culture medium was injected in the tumor tissue of control group(n=10).The maximum diameter of tumor mass was measured by ultrasound at 2,4 weeks after vx-2 tumor mass was embedded. All animals were sacrificed at 4 weeks.The double labeling immunofluorescence for CD146 was performed to reveal whether engrafted cells can differentiate into vascular endothelial cells.Vascular density was compared between two groups.VX2 cells were primary cultured also.MSCs were incubated in 30%VX2 conditioned medium for 7 or 14 days.The protein expression of CD146 were measured by Westernblot methods.RESULTS There was no significant difference in maximum diameter of tumor mass between two groups at 2 weeks(test group 0.77±0.15cm vs control group 0.71±0.15cm,P＞0.05).The maximum diameter appear larger in test group at 4weeks (test group 3.82±0.94cm vs control group 2.28±0.54cm,P＜0.05). Immunofluorescence studies revealed some engrafted MSCs expressing a vascular endothelial cell phenotype(CD146).Furthermore,vascular density was augmented in test group compared with control group(10.1±0.70/0.2mm~2 vs 8.24±0.81/0.2mm~2, P＜0.05).In vitro,the protein expression of CD146 significantly increased in the 7 days group than the control group(P＜0.05),which further increasd in the 14 days group(P＜0.05).CONCLUSION Engrafted MSCs can differentiate into vascular endothelial cells and contribute to angiogenesis in tumor microenvironment,which may be the major pathway of promoting tumor growth. OBJECTIVE To evaluate a noninvasive method to predict bladder outlet obstruction(BOO) and bladder function in patients with benign prostatic enlargement (BPE) based on intravesical prostatic protrusion(IPP) using transabdominal ultrasound.METHODS The records of 206 first visit patients with BPE were analyzed. Patients were divided into two groups based on the degree of IPP:the significant IPP group-greater than 10 mm and no significant IPP group-10 mm or less.Clinical data and urodynamic findings of the two groups were analyzed to define the clinical significance of IPP.RESULTS Increased prostate volume(73.7±35.9cm~3 vs 62.8±36.5cm~3),serum prostate specific antigen(1.81±0.67ng/ml vs 1.64±0.36ng/ml),post-voiding residual urine volume(290.2±217.2ml vs 228.2±167.9ml),incidence of acute urine residual (33.3%vs 18.0%) and bladder trabeculation(23.1%vs 11.7%) appeared more often in the significant IPP group(P＜0.05).In the urodynamic findings,significantly lower peak flow rate(Qmax)(7.6±4.1 ml/s vs 9.1±3.6ml/s ) and higher incidence of detrusor overactivity(82.1%vs 17.2%) and low bladder compliance(35.9%vs 12.5%) both existed in the significant IPP group(P＜0.01).In addition,maximum detrusor pressure (109.8±84.9cmH20 vs 84.9±44.1 cmH2O) and BOO index(75.2±27.1 vs 65.9±34.6) were significantly higher in the significant IPP group(p＜0.05).The incidence of recurrence of acute urinary intention was higher in the significant IPP group(64.3% vs 23.5%)(P＜0.05).CONCLUSION IPP is a useful predictor for evaluating BOO and detrusor function. BOO and impaired detrusor function in the significant IPP patients are more severe. The significant IPP patients,especially those presenting with AUR,may benefit from early surgical intervention.更多还原