本文選題：卵巢腫瘤 + 肝細(xì)胞生長因子�。� 參考：《遵義醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：肝細(xì)胞生長因子可以促進(jìn)細(xì)胞的分裂、再生和運(yùn)動，它及其受體c-Met異常高表達(dá)在卵巢癌進(jìn)展中起重要作用。凋亡抑制蛋白XIAP在卵巢癌的發(fā)生中也有著一定的關(guān)系，但關(guān)于三者在卵巢癌中的調(diào)控的研究目前尚未見報道。本實(shí)驗運(yùn)用免疫組織化學(xué)檢測HGF及c-Met在卵巢癌中的表達(dá)情況，再運(yùn)用c-Met的特異性抑制劑PHA665752作用于卵巢癌SKOV-3后，觀察細(xì)胞增殖情況、凋亡改變及對XIAP的影響。為卵巢癌臨床治療提供理論依據(jù)。 方法：（1）免疫組織化學(xué)染色（EnVision法）及Image-ProPlus圖像分析軟件，觀察HGF及其受體在卵巢漿液性囊腺癌組織、卵巢漿液性囊腺瘤、卵巢交界性腫瘤和正常卵巢組織中的蛋白表達(dá)。（2）以人卵巢漿液性乳頭狀囊腺癌細(xì)胞系SKOV-3為研究對象，首先在37℃，5%CO2，20%O2和95%濕度條件下培養(yǎng)卵巢癌細(xì)胞，提取生長指數(shù)期細(xì)胞，，且生長良好的細(xì)胞進(jìn)行以下實(shí)驗。以不同濃度（0ng/ml、20ng/ml、40ng/ml、80ng/ml）的HGF和（0.5umol/l、1umol/l、2umol/l）的PHA665752作用細(xì)胞后，于不同時間（24h、48h、72h）收集細(xì)胞，使用MTT法、流式細(xì)胞術(shù)AnnexinV-FITC/PI法檢測各組卵巢癌細(xì)胞的增殖及凋亡情況。（3）PT-PCR、Western Blot檢測PHA6657522umol/l、HGF40ng/ml作用細(xì)胞48小時后XIAP、HGF、c-Met的改變情況。（4）所得數(shù)據(jù)以錯誤！未找到引用源。表示，多組間均數(shù)比較采用方差分析，p0.05有統(tǒng)計學(xué)意義，統(tǒng)計使用SPSS17.0軟件處理。 結(jié)果：（1）卵巢良性腫瘤HGF、c-Met的表達(dá)水平相對于正常對照組來說，增高不明顯，差異無統(tǒng)計學(xué)意義（P＞0.05），陽性率是20％、20％；卵巢惡性腫瘤HGF、c-Met的表達(dá)水平相對于良性腫瘤來說，增高明顯，差異有統(tǒng)計學(xué)意義（P＜0.05），陽性率是60％、70％。（2）不同濃度（0ng/ml、20ng/ml、40ng/ml、80ng/ml）的HGF和（0umol/l、0.5umol/l、1umol/l、2umol/l）的PHA665752對卵巢癌存活率的影響：24h分別為（44.58%、53.77%、59.94%、63.97%）和（60.05%、57.31%、52.05%、44.58%）；48h分別為（38.48%、84.33%、84.48%、81.85%）和（57.05%、46.72%、39.28%、37.66%）；72h分別為（29.26%、30.18%、31.23%、32.28%）和（49.26%、39.28%、32.41%、34.43%）。20ng/ml HGF隨作用時間延長，細(xì)胞增殖無明顯改變，差異無統(tǒng)計學(xué)意義（p0.05）；而40ng/ml、80ng/mlHGF隨作用時間延長，細(xì)胞存活率增高，各組比較有統(tǒng)計學(xué)差異（p0.05）；當(dāng)作用24小時后隨著濃度增加，存活率無明顯改變，差異無統(tǒng)計學(xué)意義（p0.05）；當(dāng)作用時間延長48h、72h，存活率隨濃度的提高而增加，差異有統(tǒng)計學(xué)意義（p0.05）。不同濃度的PHA665752作用24h后細(xì)胞增殖活性無明顯改變，統(tǒng)計學(xué)比較無差異（p0.05）；而在48h、72h時隨著濃度的增加（1umol/l、2umol/l），細(xì)胞存活率減少，各組比較有統(tǒng)計學(xué)差異（p0.05）；0.5umol/lPHA665752隨著作用時間延長，細(xì)胞增殖活性也無明顯改變，統(tǒng)計學(xué)比較無差異（p0.05），而1umol/l、2umol/lPHA665752隨著作用時間的延長，細(xì)胞存活率下降，各組比較有統(tǒng)計學(xué)差異（p0.05）。（3）不同濃度（0ng/ml、20ng/ml、40ng/ml、80ng/ml）的HGF和（0.5umol/l、1umol/l、2umol/l）的PHA665752對卵巢癌凋亡率的影響：24h分別為（13.63%、13.27%、13.51%、13.20%）和（9.71%、10.33%、14.13%、15.61%）；48h分別為（14.37%、13.75%、9.87%、10.90%）和（10.66%、15.49%、30.61%、19.26%）；72h分別為（14.18%、12.26%、11.45%、13.76%）和（14.09%、17.45%、27.19%、20.28%）。HGF實(shí)驗組24h各濃度組間凋亡率無明顯差異，統(tǒng)計學(xué)比較無差異（p0.05），而在48h、72h隨著HGF濃度增加（40ng/ml、80ng/ml），細(xì)胞凋亡降低，統(tǒng)計學(xué)比較有差異（p0.05）。PHA665752相同濃度時，隨著作用時間的延長，細(xì)胞凋亡率增加，其差異有統(tǒng)計學(xué)意義（p0.05）；0.5umol/lPHA665752作用24h后細(xì)胞凋亡率無明顯改變，差異無統(tǒng)計學(xué)意義（p0.05），而在48h、72h是隨著濃度增加1umol/l、2umol/l，凋亡率增加，其差異有統(tǒng)計學(xué)意義（p0.05）。（4）空白對照組HGF mRNA相對表達(dá)含量為0.88±0.11、c-Met mRNA相對表達(dá)含量為0.85±0.14、XIAP mRNA相對表達(dá)含量為0.92±0.08，實(shí)驗組HGF40ng/ml對卵巢癌SKOV3作用48h后，HGF mRNA相對表達(dá)含量為1.32±0.25、c-Met mRNA相對表達(dá)含量為1.69±0.16、XIAP mRNA相對表達(dá)含量為1.92±0.07；PHA6657521umol/l對卵巢癌SKOV3作用48h后，HGF mRNA相對表達(dá)含量為0.71±0.16、c-Met mRNA相對表達(dá)含量為0.49±0.05、XIAP mRNA相對表達(dá)含量為0.42±0.02。PHA665752處理組中各蛋白比空白組相對表達(dá)量低，其差異有統(tǒng)計學(xué)意義（p0.05），HGF處理組中各蛋白比空白組中相對表達(dá)量高，其差異有統(tǒng)計學(xué)意義（p0.05）。（5）空白對照組XIAP蛋白表達(dá)量0.70±0.05，HGF處理組XIAP蛋白表達(dá)量0.99±0.03，PHA665752處理組XIAP蛋白表達(dá)0.34±0.03。HGF處理組XIAP蛋白表達(dá)量比空白組高，其差異有統(tǒng)計學(xué)意義（p0.05）、PHA665752處理組XIAP蛋白表達(dá)量比空白組低，其差異有統(tǒng)計學(xué)意義（p0.05）�？瞻讓φ战Mc-Met蛋白表達(dá)量0.69±0.03，HGF處理組c-Met蛋白表達(dá)量1.00±0.03，PHA665752處理組c-Met蛋白表達(dá)0.35±0.05。HGF處理組c-Met蛋白表達(dá)量比空白組高，其差異有統(tǒng)計學(xué)意義（p0.05）、PHA665752處理組c-Met蛋白表達(dá)量比空白組低，其差異有統(tǒng)計學(xué)意義（p0.05）。 結(jié)論：（1）HGF、c-Met、XIAP在惡性卵巢癌中高表達(dá)。（2）PHA665752可抑制c-Met，可使HGF/c-Met信號通路下調(diào)，從而使HGFmRNA、c-MetmRNA、XIAPmRNA表達(dá)減少，蛋白表達(dá)量也減少，最終導(dǎo)致卵巢癌SKOV3細(xì)胞增殖減少，凋亡增加。（3）HGF能使HGFmRNA、c-MetmRNA、XIAPmRNA表達(dá)增加，蛋白表達(dá)量也增加，促進(jìn)卵巢癌SKOV3細(xì)胞生長�？梢�，卵巢癌SKOV3細(xì)胞中HGF、c-Met、XIAP表達(dá)水平受HGF/c-Met通路的調(diào)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.

【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.31

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