c-Met抑制剂PHA665752对卵巢癌细胞凋亡影响的实验研究

发布时间：2018-04-23 02:00

本文选题：卵巢肿瘤 + 肝细胞生长因子　；参考：《遵义医学院》2014年硕士论文

【摘要】：目的：肝细胞生长因子可以促进细胞的分裂、再生和运动，它及其受体c-Met异常高表达在卵巢癌进展中起重要作用。凋亡抑制蛋白XIAP在卵巢癌的发生中也有着一定的关系，但关于三者在卵巢癌中的调控的研究目前尚未见报道。本实验运用免疫组织化学检测HGF及c-Met在卵巢癌中的表达情况，再运用c-Met的特异性抑制剂PHA665752作用于卵巢癌SKOV-3后，观察细胞增殖情况、凋亡改变及对XIAP的影响。为卵巢癌临床治疗提供理论依据。方法：（1）免疫组织化学染色（EnVision法）及Image-ProPlus图像分析软件，观察HGF及其受体在卵巢浆液性囊腺癌组织、卵巢浆液性囊腺瘤、卵巢交界性肿瘤和正常卵巢组织中的蛋白表达。（2）以人卵巢浆液性乳头状囊腺癌细胞系SKOV-3为研究对象，首先在37℃，5%CO2，20%O2和95%湿度条件下培养卵巢癌细胞，提取生长指数期细胞，，且生长良好的细胞进行以下实验。以不同浓度（0ng/ml、20ng/ml、40ng/ml、80ng/ml）的HGF和（0.5umol/l、1umol/l、2umol/l）的PHA665752作用细胞后，于不同时间（24h、48h、72h）收集细胞，使用MTT法、流式细胞术AnnexinV-FITC/PI法检测各组卵巢癌细胞的增殖及凋亡情况。（3）PT-PCR、Western Blot检测PHA6657522umol/l、HGF40ng/ml作用细胞48小时后XIAP、HGF、c-Met的改变情况。（4）所得数据以错误！未找到引用源。表示，多组间均数比较采用方差分析，p0.05有统计学意义，统计使用SPSS17.0软件处理。结果：（1）卵巢良性肿瘤HGF、c-Met的表达水平相对于正常对照组来说，增高不明显，差异无统计学意义（P＞0.05），阳性率是20％、20％；卵巢恶性肿瘤HGF、c-Met的表达水平相对于良性肿瘤来说，增高明显，差异有统计学意义（P＜0.05），阳性率是60％、70％。（2）不同浓度（0ng/ml、20ng/ml、40ng/ml、80ng/ml）的HGF和（0umol/l、0.5umol/l、1umol/l、2umol/l）的PHA665752对卵巢癌存活率的影响：24h分别为（44.58%、53.77%、59.94%、63.97%）和（60.05%、57.31%、52.05%、44.58%）；48h分别为（38.48%、84.33%、84.48%、81.85%）和（57.05%、46.72%、39.28%、37.66%）；72h分别为（29.26%、30.18%、31.23%、32.28%）和（49.26%、39.28%、32.41%、34.43%）。20ng/ml HGF随作用时间延长，细胞增殖无明显改变，差异无统计学意义（p0.05）；而40ng/ml、80ng/mlHGF随作用时间延长，细胞存活率增高，各组比较有统计学差异（p0.05）；当作用24小时后随着浓度增加，存活率无明显改变，差异无统计学意义（p0.05）；当作用时间延长48h、72h，存活率随浓度的提高而增加，差异有统计学意义（p0.05）。不同浓度的PHA665752作用24h后细胞增殖活性无明显改变，统计学比较无差异（p0.05）；而在48h、72h时随着浓度的增加（1umol/l、2umol/l），细胞存活率减少，各组比较有统计学差异（p0.05）；0.5umol/lPHA665752随着作用时间延长，细胞增殖活性也无明显改变，统计学比较无差异（p0.05），而1umol/l、2umol/lPHA665752随着作用时间的延长，细胞存活率下降，各组比较有统计学差异（p0.05）。（3）不同浓度（0ng/ml、20ng/ml、40ng/ml、80ng/ml）的HGF和（0.5umol/l、1umol/l、2umol/l）的PHA665752对卵巢癌凋亡率的影响：24h分别为（13.63%、13.27%、13.51%、13.20%）和（9.71%、10.33%、14.13%、15.61%）；48h分别为（14.37%、13.75%、9.87%、10.90%）和（10.66%、15.49%、30.61%、19.26%）；72h分别为（14.18%、12.26%、11.45%、13.76%）和（14.09%、17.45%、27.19%、20.28%）。HGF实验组24h各浓度组间凋亡率无明显差异，统计学比较无差异（p0.05），而在48h、72h随着HGF浓度增加（40ng/ml、80ng/ml），细胞凋亡降低，统计学比较有差异（p0.05）。PHA665752相同浓度时，随着作用时间的延长，细胞凋亡率增加，其差异有统计学意义（p0.05）；0.5umol/lPHA665752作用24h后细胞凋亡率无明显改变，差异无统计学意义（p0.05），而在48h、72h是随着浓度增加1umol/l、2umol/l，凋亡率增加，其差异有统计学意义（p0.05）。（4）空白对照组HGF mRNA相对表达含量为0.88±0.11、c-Met mRNA相对表达含量为0.85±0.14、XIAP mRNA相对表达含量为0.92±0.08，实验组HGF40ng/ml对卵巢癌SKOV3作用48h后，HGF mRNA相对表达含量为1.32±0.25、c-Met mRNA相对表达含量为1.69±0.16、XIAP mRNA相对表达含量为1.92±0.07；PHA6657521umol/l对卵巢癌SKOV3作用48h后，HGF mRNA相对表达含量为0.71±0.16、c-Met mRNA相对表达含量为0.49±0.05、XIAP mRNA相对表达含量为0.42±0.02。PHA665752处理组中各蛋白比空白组相对表达量低，其差异有统计学意义（p0.05），HGF处理组中各蛋白比空白组中相对表达量高，其差异有统计学意义（p0.05）。（5）空白对照组XIAP蛋白表达量0.70±0.05，HGF处理组XIAP蛋白表达量0.99±0.03，PHA665752处理组XIAP蛋白表达0.34±0.03。HGF处理组XIAP蛋白表达量比空白组高，其差异有统计学意义（p0.05）、PHA665752处理组XIAP蛋白表达量比空白组低，其差异有统计学意义（p0.05）。空白对照组c-Met蛋白表达量0.69±0.03，HGF处理组c-Met蛋白表达量1.00±0.03，PHA665752处理组c-Met蛋白表达0.35±0.05。HGF处理组c-Met蛋白表达量比空白组高，其差异有统计学意义（p0.05）、PHA665752处理组c-Met蛋白表达量比空白组低，其差异有统计学意义（p0.05）。结论：（1）HGF、c-Met、XIAP在恶性卵巢癌中高表达。（2）PHA665752可抑制c-Met，可使HGF/c-Met信号通路下调，从而使HGFmRNA、c-MetmRNA、XIAPmRNA表达减少，蛋白表达量也减少，最终导致卵巢癌SKOV3细胞增殖减少，凋亡增加。（3）HGF能使HGFmRNA、c-MetmRNA、XIAPmRNA表达增加，蛋白表达量也增加，促进卵巢癌SKOV3细胞生长。可见，卵巢癌SKOV3细胞中HGF、c-Met、XIAP表达水平受HGF/c-Met通路的调控。
[Abstract]:Objective: hepatocyte growth factor (hepatocyte growth factor) can promote cell division, regeneration and movement, and its abnormal high expression of c-Met plays an important role in the progression of ovarian cancer. Apoptosis suppressor protein XIAP also has a certain relationship in the occurrence of ovarian cancer, but the study of the regulation of the three in ovarian cancer has not yet been reported. The expression of HGF and c-Met in ovarian cancer was detected by immunohistochemistry, and the effect of c-Met specific inhibitor PHA665752 on ovarian cancer SKOV-3 was used to observe the cell proliferation, apoptosis and the effect on XIAP. The theoretical basis for the clinical treatment of ovarian cancer was provided.
Methods: (1) immunohistochemical staining (EnVision) and Image-ProPlus image analysis software were used to observe the protein expression of HGF and its receptor in ovarian serous cystadenocarcinoma, ovarian serous cystadenoma, borderline ovarian tumor and normal ovarian tissue. (2) human ovarian serous papillary cystadenocarcinoma cell line SKOV-3 as the object of study. Firstly, the ovarian cancer cells were cultured at 37 degrees, 5%CO2,20%O2 and 95%, and the growth index cells were extracted, and the cells with good growth were carried out in the following experiments. The cells were collected at different concentrations (0ng/ml, 20ng/ml, 40ng/ml, 80ng/ml) HGF and PHA665752 (0.5umol/l, 1umol/l, 2umol/l) at different time (24h, 48h, and 2umol/l). TT method, flow cytometry AnnexinV-FITC/PI method to detect the proliferation and apoptosis of ovarian cancer cells in each group. (3) PT-PCR, Western Blot detection PHA6657522umol/l, HGF40ng/ml after 48 hours XIAP, HGF, c-Met changes. (4) the data were wrong! No reference source was found. Statistical significance, statistical use of SPSS17.0 software processing.
Results: (1) the expression level of HGF and c-Met was not significantly higher than that of the normal control group (P > 0.05), and the positive rate was 20%, 20%. The expression level of ovarian malignant tumor was HGF, and the expression level of c-Met was higher than that of benign tumor (P < 0.05), the positive rate was 60%, 7. 0%. (2) HGF and PHA665752 on the survival of ovarian cancer (0umol/l, 0.5umol/l, 1umol/l, 2umol/l) of different concentrations (0ng/ml, 20ng/ml, 40ng/ml, 80ng/ml): 24h (44.58%, 53.77%, 59.94%, 63.97%) and (60.05%, 57.31%, 52.05%, 44.58%) and (38.48%, 84.33%, 84.48%, 81.85%) and (38.48%, 38.48%, 38.48%); Do not (29.26%, 30.18%, 31.23%, 32.28%) and (49.26%, 39.28%, 32.41%, 34.43%).20ng/ml HGF with time prolonged, no significant changes in cell proliferation, the difference was not statistically significant (P0.05), while 40ng/ml, 80ng/mlHGF increased with the time of action, the cell survival rate increased, and there was a statistically significant difference (P0.05) in each group (P0.05) after 24 hours. There was no significant change in the survival rate, the difference was not statistically significant (P0.05). When the action time extended 48h, 72h, the survival rate increased with the increase of concentration, and the difference was statistically significant (P0.05). There was no significant change in cell proliferation activity after 24h at different concentrations of PHA665752 (P0.05), but in 48h and 72h with concentration. Increase (1umol/l, 2umol/l), cell survival rate decreased, there was a statistical difference in each group (P0.05); 0.5umol/lPHA665752 had no significant changes in cell proliferation activity with the prolongation of action time (P0.05), but 1umol/l, 2umol/lPHA665752 decreased with the prolongation of action time, and there was a statistical difference in each group. P0.05. (3) the effect of HGF and PHA665752 (0.5umol/l, 1umol/l, 2umol/l) on the apoptosis rate of ovarian cancer in different concentrations (0ng/ml, 20ng/ml, 40ng/ml, 80ng/ml) and 24h were (13.63%, 13.27%, 13.51%, 13.20%) and (9.71%, 10.33%, 14.13%, 15.61%), respectively; 48h (14.37%, 13.75%, 9.87%, 10.90%) and (14.37%, 9.87%, 10.90%), respectively; There was no significant difference in the apoptosis rate between the groups of.HGF experimental group (14.18%, 12.26%, 11.45%, 13.76%) and (14.09%, 17.45%, 27.19%, 20.28%), and there was no difference between the groups (P0.05), but in 48h, 72h with the increase of HGF concentration (40ng/ml, 80ng/ml), the apoptosis decreased, and the statistical difference (P0.05).PHA665752 same concentration, along with the effect. The apoptosis rate of cells increased, the difference was statistically significant (P0.05), and there was no significant change in the apoptosis rate of 0.5umol/lPHA665752 after 24h (P0.05), but in 48h, 72h was increased with the concentration of 1umol/l, 2umol/l, and the difference was statistically significant (P0.05). (4) HGF mRNA phase in the blank control group. The expression content was 0.88 + 0.11, the relative expression content of c-Met mRNA was 0.85 + 0.14, and the relative expression content of XIAP mRNA was 0.92 + 0.08. The relative expression content of HGF mRNA was 1.32 + 0.25 and the relative expression content of c-Met mRNA was 1.69 + 0.16 after 48h in the experimental group of ovarian cancer, and the relative expression content of XIAP c-Met was 1.92 + 0.07. The relative expression of HGF mRNA was 0.71 + 0.16, the relative expression content of c-Met mRNA was 0.49 + 0.05, and the relative expression of XIAP mRNA relative expression was lower than that of the blank group. The difference was statistically significant (P0.05), and the proteins in the HGF treatment group were relative to the blank group after 48h. The relative expression of c-Met mRNA was 0.71 +. The difference was statistically significant (P0.05). (5) the expression of XIAP protein in the blank control group was 0.70 + 0.05, the expression of XIAP protein in the HGF treatment group was 0.99 + 0.03. The XIAP protein expression of the XIAP protein expression group in the PHA665752 treatment group was higher than that in the blank group, and the difference was statistically significant (P0.05), and the XIAP protein expression in the PHA665752 treatment group was expressed. The difference was significantly lower than that in the blank group (P0.05). The expression of c-Met protein in the blank control group was 0.69 + 0.03, the expression of c-Met protein in the HGF treatment group was 1 + 0.03. The c-Met protein expression of the c-Met protein expression group in the PHA665752 treatment group was higher than that in the blank group, and the difference was statistically significant (P0.05), PHA665752 treatment group c-Met eggs The white expression level was lower than that in the blank group, and the difference was statistically significant (P0.05).
Conclusion: (1) HGF, c-Met and XIAP are highly expressed in malignant ovarian cancer. (2) PHA665752 can inhibit c-Met, which can reduce the HGF/c-Met signaling pathway, thus reducing the expression of HGFmRNA, c-MetmRNA, XIAPmRNA and protein expression, which eventually leads to the decrease in proliferation of SKOV3 cells and the increase of apoptosis in ovarian cancer SKOV3 cells. (3) HGF can make HGFmRNA, HGFmRNA, and expression increase The protein expression level also increased, which promoted the growth of ovarian cancer SKOV3 cells. Therefore, the expression level of HGF, c-Met and XIAP in ovarian cancer SKOV3 cells was regulated by HGF/c-Met pathway.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】