【作者】 陈伟；

【导师】 曹杰； 唐伟镖；

【作者基本信息】 广州医学院 ， 胃肠外科， 2009， 硕士

【摘要】 背景近年来结肠癌发病率和死亡率均呈上升趋势。手术仍是治疗的关键手段,而术后放、化疗对提高生存率作用有限。因此,寻求新的结肠癌早期诊断和治疗的方法对于提高其手术切除率和生存率,改善生活质量具有重要的意义。随着肿瘤免疫学和分子生物学研究的进展,结肠癌发生发展和转移的分子机制已经部份阐明,为结肠癌的基因治疗提供了理论基础。本课题组的前期研究证明脆性组氨酸三联体(Fragile histidine triad,FHIT)基因表达缺失在结肠癌的发生、发展中起着重要的作用,但目前其应用于基因治疗总的效果仍不理想,许多还处于临床前实验阶段,加上人体基因表达机制十分复杂,到目前为止有些环节还不清楚,需要继续深入研究,以便更好地发挥基因治疗的作用。外源性FHIT基因转染入FHIT基因表达缺失的结肠癌中诱导癌细胞凋亡的分子机制一旦明确,将为结肠癌的治疗提供全新的分子治疗策略。本研究拟通过阳离子脂质体Lipofectamine 2000的介导下将基因工程技术构建的真核表达载体pFHIT转染入人结肠癌细胞系SW480中,通过G418筛选、滤纸片法结合有限稀释法挑选单克隆稳定转染株,检测SW480-FHIT、转染空质粒pRc/CMV2的SW480-NEO细胞株以及野生型SW480细胞系的FHIT表达情况;观察FHIT在体外对细胞生长增殖及细胞周期和凋亡的影响;建立BALB/C NU裸鼠SW480皮下结肠癌模型,探讨FHIT在体内抗肿瘤效应。第一部分构建稳定表达FHIT的SW480结肠癌细胞株一.目的构建稳定表达FHIT的SW480结肠癌细胞株。二.方法1.细胞培养SW480细胞复苏后以含10%胎牛血清、高糖DMEM完全培养基常规传代培养。2.真核表达载体的转染、G418筛选及挑选单克隆稳定转染株以阳离子脂质体Lipofectamine 2000介导将FHIT表达载体质粒pFHIT(由本课题组杨平师兄构建)和空载体质粒pRc/CMV2转染人结肠癌细胞株SW480。转染48小时后,采用梯度渐增法确定的含G418完全DMEM培养基对转染细胞进行筛选。维持最适筛选浓度14天后用滤纸消化法结合有限稀释法挑选并扩增单克隆细胞株。3. RT-PCR及Western Blot检测各组细胞FHIT表达的检测Trizol法提取细胞总RNA,样品进行紫外分光光度仪检测及1%琼脂糖胶电泳检测判断产量及质量。RT-PCR检测FHIT表达。PLC蛋白裂解液裂解细胞后提取总蛋白,行SDS-PAGE电泳,转膜后孵育1抗、2抗,ECL发光法检测目的FHIT蛋白表达。三.结果1.重组质粒的鉴定结果分别以FHIT重组质粒以及pRc/CMV2质粒为模板,加入FHIT引物构建PCR体系进行特异性扩增,产物进行1%琼脂糖凝胶电泳,可见FHIT为模板组出现431bp的条带,而pRc/CMV2则无相应条带。2.真核载体转染、G418筛选、有限稀释法挑选单克隆稳定转染株结果使用Lipofectamine 2000阳离子脂质体进行真核细胞载体的转染对细胞的损伤较小,转染效果满意。对SW480细胞系来说,含800μg/ml G418的完全DMEM培养基为最佳筛选浓度,400μg/ml为合理维持浓度。从转染成功并通过筛选的细胞中通过滤纸消化法和有限稀释法挑选单克隆并建立稳定表达细胞株。3.稳定转染株FHIT表达评价分别以FHIT重组质粒以及pRc/CMV2质粒和空白SW480细胞株cDNA为模板,进行RT-PCR,转染FHIT重组质粒的实验组样品可扩增出FHIT产物条带,空白对照组、阴性对照组样品均未扩增出FHIT产物条带。而相应的Western blot检测转染FHIT的细胞中有FHIT蛋白表达,而转染空载体的细胞株和阴性对照均无目的条带出现。三组均可扩增出内参GAPDH条带。四.结论使用Lipofectamine 2000阳离子脂质体进行真核细胞载体的转染可以得到满意的转染效果。通过G418筛选、滤纸法结合有限稀释法挑选单克隆,可以建立FHIT重组质粒稳定转染的人结肠癌细胞株。转染了pFHIT的人结肠癌SW480细胞株可以稳定表达FHIT,转染空质粒pRc/CMV2及未转染质粒的SW480野生型细胞均不表达FHIT。第二部分FHIT对SW480人结肠癌细胞生长增殖影响的体外实验一目的:通过MTT法检测细胞增殖能力、流式细胞仪分析细胞周期和凋亡改变,进而分析FHIT基因转染对SW480人结肠癌细胞生长增殖的影响。二.方法1.分组:挑选稳定表达FHIT的转染株(设定为SW480-FHIT)进行以下实验;挑选不表达FHIT的pRc/CMV2转染株(设定为SW480-NEO)为阴性对照组;以未转染的SW480为空白对照组。2.观察细胞形态使用光学显微镜观察各组细胞形态。3. MTT检测细胞增殖能力将上述三组细胞以每孔1×104个细胞量接种到96孔板,每组设置4个复孔,接种细胞后于不同的时间点采用MTT法检测细胞增殖能力改变,并绘制细胞生长曲线。4.流式细胞仪分析细胞周期和凋亡于6孔板内接种上述三组细胞,待细胞长至90%汇合度时,胰酶消化法收取细胞,调整细胞密度为1×106/ml,PI染色,以流式细胞仪检测细胞周期和凋亡变化,每组重复3次。三.结果1.细胞生长情况正常营养、同等培养条件下,3组细胞的生长状态和形态观察显示无明显差异。2、噻唑蓝比色法(MTT)检测转染前后细胞增殖能力的变化空载体转染细胞SW480-Neo和亲本细胞SW480增殖无显著差异(P>0.05),相比之下,FHIT基因转染的细胞株SW480-FHIT较上述两组细胞生长速度明显减慢,尤其是达到对数生长期后更加明显,差异显著(P<0.05),。3、SW480细胞转染FHIT前后细胞周期和凋亡比率的变化: SW480-Neo和SW480细胞的细胞周期和凋亡比率检测均未见显著差异,而SW480-FHIT细胞和上述两组细胞对比,G2/M期和S期比例显著下降,而G0/G1期的细胞比例显著增加,且凋亡率高于较其它两组(P<0.05)。四.结论FHIT基因可以降低人结肠癌细胞SW480的增殖能力,抑制其细胞周期并增加细胞的凋亡率。第三部分FHIT对SW480人结肠癌细胞生长增殖影响的体内实验一、目的FHIT基因转染对SW480人结肠癌细胞在裸鼠体内的生长增殖影响,并对其机制进行初步探讨。二、方法1.实验动物分组60只SPF级4～5周龄雄性BALB/C NU裸鼠,随机分为三组,每组20只,实验组接种SW480-FHIT,阴性对照组接种SW480-NEO,空白对照组接种SW480。2.动物模型的建立常规培养细胞数量足够并生长至对数期时,收集细胞镜下计数,PBS重悬细胞制备单细胞悬液,使活细胞浓度为5×107/ml。每只BALB/C NU裸鼠以0.2ml(即1×107个细胞)接种于右侧背部皮下。3.实验观察观察肿瘤形成情况;接种后第10天开始隔2天测量肿瘤体积,并绘制各组肿瘤生长曲线。肿瘤体积(mm3)以公式0.5×a×b2计算,a(mm)为肿瘤长径,b(mm)为肿瘤短径;待肿瘤体积至500mm3左右时每组随机选取10只小鼠处死用于检测,其余10只继续培养,记录生存时间,比较三组荷瘤小鼠的生存期。4.肿瘤标本检测内容:(1) RT-PCR和Western Blot检测肿瘤组织中FHIT表达。(2)免疫组化检测肿瘤组织FHIT、P53基因表达。三、结果1. FHIT对BALB/C NU裸鼠移植瘤生长的影响在接种后7～10天,三组BALB/C NU裸鼠均可于皮下触及肿瘤。肿瘤生长曲线可见SW480-FHIT组的肿瘤生长比SW480组和SW480-NEO组的肿瘤生长明显为慢,对各时间点的肿瘤体积进行单因素方差分析,比较组间差异有统计学意义(SW480-FHIT组和SW480相比,各时间点p<0.01, SW480-FHIT组和SW480-NEO相比,各时间点p<0.01)。而两对照组间的差异没有统计学意义(各时间点p>0.05)。2. FHIT对BALB/C NU荷瘤裸鼠生存期的影响SW480组的中位生存期为35天,95%CI(30.868,39.132);SW480-NEO组为37天, 95%CI(35.482,38.518);SW480-FHIT为48天,95%CI (43.868,52.132),三组的平均生存期分别为34.600±1.558、36.100±1.479、50.700±3.685天。Log Rank法分析组间差异,见两对照组间生存期差异无统计学意义(p=0.474),而实验组分别与两对照组相比,差异均有统计学意义(p<0.001)。3.肿瘤组织FHIT基因的表达情况RT-PCR检测结果提示SW480-FHIT组小鼠肿瘤组织中均有不同程度的FHIT基因表达,而Western Blot则表明FHIT蛋白有表达;相比之下,其它各组小鼠mRNA和蛋白水平均未检测到FHIT表达。免疫组化结果显示,SW480-FHIT组小鼠肿瘤组织中有FHIT蛋白表达,而其它两组无表达;P53结果分析显示,各组均有P53表达,但以SW480-FHIT组最为显著。四、结论1、FHIT基因转染结肠癌细胞能显著抑制肿瘤细胞在体内的生长、延长荷瘤裸鼠的生存期;2、FHIT基因转染的SW480细胞接种后形成的肿瘤组织中有FHIT表达,且P53基因表达增加,说明FHIT有可能通过上调P53基因的表达抑制了肿瘤细胞的增殖。更多还原

【Abstract】 Colon Carcinoma becomes more and more commonly seen and the death caused by it is increasing worldwide recently. Surgery is the most important treatment method but chemotherapy failed to significantly benefit the patient after the surgery. Therefore, in order to increase survival rate and improve life quality, searching for method of early diagnosis and treatment is of most importance. Recent development in tumor immunology and molecular biology has shed some light on the mechanism of the occurrence and transferring of the Colon Carcinoma, which provides basis to gene therapy. Preliminary experiments have shown that the failed expression of Fragile Histidine Triad (FHIT) play an important role in the initiation and development of the Colon Carcinoma, but its application in gene therapy is still under satisfaction; many of them are still under clinical experiment phase. Moreover, due to the complication of the expression of the human genes, in-depth research is needed to elucidate some unknown factors, in order to fully utilize the advantage of the gene therapy. Once the mechanism of the cancer cell death due to transfection of FHIT into Colon Carcinoma cells is elucidated, new strategies of gene therapy of Colon Carcinoma can be formulated.This research aim to employ cationic liposome Lipofectamine 2000 to trasfect eukaryotic vector pFHIT, obtained through gene engineering, into Human Colon Carcinoma cell series SW480. Via G418 screening, paper disc method combined with optimal concentration, we can select the stable mono cloned transfected gene, test for SW480-FHIT, explore the degree of expression of FHIT in SW480-NEO cells after transfection of pRc/CMV2, as well as in wild type SW480 cells. We also observe the effect of FHIT on cell growth in vitro, establish the SW480 Colon Carcinoma pathological model in BALB/C NU naked mouse, and discuss the antitumor effect of FHIT.PartⅠUtilization of Gene Transfection to Establish Stable SW480 Transfectant of FHITObjectiveUtilization of Gene Transfection to Establish Stable SW480 Transfectant of FHITMethod1. Cell culture SW480 Colon Carcinoma cells were maintained in complete high glucose DMEM medium containing 10% fetal bovine serum and 2mM L-Glutamine.2. Gene transfer into cells, G418 selection and Clone of the stable transfectants SW480 Colon Carcinoma cells were transfected with pFHIT (constructed by former group member Ping Yang) and pRc/CMV2 via cationic liposome Lipofectamine 2000. After 48 hours, a series of complete DMEM dedium with gradient elevated concentration of G418 was used to culture and select the transfected cells for three to four weeks, including a two week selection period with optimal concentration of G418. G418-resistant Colon Carcinoma cells were then cloned via optimal concentration method.3. PT-PCR and Western Blot for detecting FHIT expression Total RNA was extracted from all the stable transfectants and their parental wild type Colon Carcinoma cells using TRIzol reagent. UV absorbance and 1% formaldehyde denatured gel electrophoresis were done. And expression of the protein was detected by Western Blotting.Result1. Identification of the recombinant plasmid Use FHIT primer to detect FHIT fragment. PCR products were run on a 1% agarose gel stained with Goldview and visualized by UV illumination. Target band showed up at around 431bp in the FHIT lane, while the parental pRc/CMV2 had no such band.2. Gene transfer into Colon Carcinoma cells, G418 selection and clone of the stable transfectantsTransfection through Lipofectamine 2000 can achieve satisfied outcome with a mild disturbance to cells. Complete DMEM containing 800μg/ml G418 is optimal for screening transfected Colon Carcinoma cells. Through G418 selection and limiting dilution of resistant cells, stable transfectants were cloned.3. FHIT expression evaluationUsing FHIT reconstructed plasmid, pRc/CMV2 and cDNA in blank SW480 as primer to run RT-PCR. The sample transfected with FHIT reconstructed plasmid shows FHIT band, whereas the blank and control experiments do not. Western blot has detected FHIT expression in FHIT transfected cells, whereas the blank and controls cells showed negative results.ConclusionTransfection through Lipofectamine 2000 can achieve satisfactory outcome with a mild disturbance to cells. Through G418 selection and limiting dilution of resistant cells, stable FHIT transfectants can be cloned. These transfectants can express FHIT whereas the vector control transfectants and the parental cell line have no FHIT expression. Part II In Vitro Proliferation Study of FHIT Expressing Transfectant MethodsObjectiveDetermination of effects of FHIT towards growth of SW480 Human Colon Carcinoma cells in votro by MTT and flow cytometer.Method1. Grouping For each stable transfectant category, one cell line was selected and named as experimental SW480-FHIT (FHIT transfected) and vector control SW480-NEO (pRc/CMV2 transfected). Parental SW480 cells were used as blank control.2. MicroscopyThree groups of cells were observed for their morphological changes under light microscopy.3. In vitro proliferation assay and cell growth chart 1×104 cells were plated into each well on the 24-well plates in triplicate and cultured for 7 days. Every 24h after seeding 3 wells of cells were collected and counted, the average number of live cells was recorded. The cell growth curve was then charted. Differences among average cell number of each group each day were compared through one-way ANOVA analysis using a SPSS13.0 software package.4. Flow cytometry (FCM) was used to detect the cell apoptosis and cycle.The cell were plated into 6-well plates, and collected when confluensing to 90%. After modulating the cell density to 1×106/ml and coloured by PI, use flow cytometry to detect the cell apoptosis and cycle.Result1. Cell growth by microscopyUnder normal culture condition, three groups of cells have no significant differences in their morphology and growth pattern, as determined by microscopy.2. Cell growth before and after FHIT transfection by MTTFHIT transfected cells showed noticeable decrease in growth rate, compared to SW480-NEO and parental SW480. This decreasing in growth rate is even more significant when the cell growth reaches exponential increasing phase (P<0.05).3. Cell cycle and death ratio of SW480 before and after FHIT transfection SW480-FHIT has significantly lower ratio of G2/M phase and S phase; more cells were in G0/G1 phase. Moreover, the death rate is higher than the rest of the two groups (P<0.05)Conclusion FHIT can significantly reduce the growth of the Human Colon Carcinoma cells, suppress their growth cycle and increase their apoptosis rate.PART III In Vivo Study of the Antitumor Function of FHIT MethodsObjectiveDetermination of effects of FHIT towards growth of SW480 Human Colon Carcinoma cells in naked mouse. Preliminary experiments towards elucidating the suppression mechanism.Method1. Grouping of animals 60 BALB/C NU mice (male, 4 to 5 weeks) were purchased and equally randomized into 3 groups. Each group received SW480-FHIT, SW480-NEO and SW480 inoculation, respectively, as it was named.2. Establishing animal model of tumor-bearing Three cell lines were cultured to the log phase with sufficient cell number, then harvested and resuspended to 5×107cells/ml in PBS. Each mouse was inoculated with 0.2ml of cell suspension (1×107 cells) subcutaneous in the right flank.3. Observation of established tumorFormation and growth of the tumor was observed. The longest (a) and shortest (b) diameters of the established tumor were measured with a caliper from the 10th day after inoculation and repeated every 2 days. The tumor volume was calculated as 0.5×a(mm)×b2(mm). Ten randomly selected mice were executed and tested when tumor volume had reached 500 mm3; the rest were kept alive and used for recording the survival time, in order to compare the life time of the tumor bearing mice. Statistical analysis was carried out with a SPSS13.0 software package.4. Detection of tumor sample.The tumors of BALB/C NU mice were sampled. Expression of FHIT mRNA was detected by RT-PCR, and expression of the protein was detected by Western Blotting.Results1. The effect of FHIT on tumor transplanted in BALB/C NU mice Three groups of mice all formed palpable subcutaneous tumor in 7~10 days after inoculation. SW480-FHIT tumor grew slower than the tumors from those two control groups according to the tumor growth curve. Differences were statistically significant compared by one-way ANOVA analysis (SW480-FHIT vs. SW480: p<0.01; SW480-FHIT vs. SW380-NEO: p<0.01), whereas tumors from the two control groups grew at about the same rate (p>0.05).2. The effect of FHIT on life time of the tumor bearing miceThe life time of SW480 group was 35 days, 95% CI (30.868,39.132); the life time of SW480-NEO group was 37 days, 95% CI (35.482, 38.518); the life time of SW480-FHIT group was 48 days, 95% CI (43.868, 52.132). The average life times for the afore-mentioned three groups were 34.600±1.558, 36.100±1.479 and 50.700±3.685 days, respectively. The difference between the experimental group and the control group were statistically meaningful (p<0.01). 3. FHIT expression in tumorRT-PCR results showed that the tumor cells in SW480-FHIT group have varies amount of FHIT expression. Western Blot had confirmed that the FHIT protein was expressed. In contrast, mRNA and protein level test were FHIT negative in the controls groups. P53 analysis showed that SW480-FHIT group has the highest P53 expression compared to the control groupsConclusion1. FHIT can significantly suppress the growth of the tumor cells in vivo and elongate the survival time of the tumor-bearing mice.2. FHIT transfected SW480 cells showed FHIT expression, and P53 expression was significantly increased, which implies that FHIT suppress growth of tumor cells by increasing the expression level of P53.更多还原