| 摘要: | I.
根據統計調查及文獻研究，發現男性罹患肝癌比率要比女性高出許多倍，除了生活形態上的不同所致，荷爾蒙的不同可能是主要原因；但是其中的機制未明仍要更進一步討論及研究。另外，在動物實驗中亦曾發現雌性素能抑制肝癌的產生。因此，在實驗中，想藉由雌性素及雌性素類似物DES、Tamoxifen及Genestine處理肝癌細胞株Hep3B，是否會促使肝癌細胞程序性的凋亡。在實驗之中，以10-6M及10-8M的劑量處理肝癌細胞株Hep3B，並透過DNA斷裂、DAPI及利用西方點墨法看細胞色素C的表現，判斷肝癌細胞株Hep3B是否有進行細胞程序性的凋亡，並利用MTT的實驗，觀察對Hep3B的存活率，及這些類似物對GST酵素活性的影響；此外，更以基因轉殖的技術轉殖野生型及dominant negative型的雌性素接受體α，對Hep3B的影響。由結果得知，雌性素及雌性素類似物對Hep3B皆會進行細胞程序性的凋亡，且比控制組明顯，另外在DAPI的實驗之中發現核濃染及核裂要比控制組來的高，而MTT的實驗中則發現在10-6M處理下，雌性素、Tamoxifen及Genistein能夠使 Hep3B的存活率下降，另外這四組中在10-6M處理下，雌性素及Genistein其GST酵素活性有明顯下降的情形發生。而在轉殖野生型的雌性素接受體的Hep3B則可發現和細胞增殖有關的轉錄因子STAT 3有明顯下降的情形，而dominant negative雌性素接受體則使STAT 3明顯上升。綜合以上結果，結論是雌性素及雌性素類似物DES、Tamoxifen及Genestine均會使Hep3B進行細胞程序性調亡，且以雌性素、Tamoxifen及Genistein對於抑制Hep3B的增殖效果較好。此外，在有野生型雌性素接受體α的情形下對於抑制Hep3B增殖效果較好。所以，在有雌性素及野生型雌性素接受體α的情形下，對肝細胞癌有較好的抑制效果。
II.
肝癌的形成導因於累積了許多和基因相關的改變，包括一些致癌基因的活化 ，或由B型肝炎病毒造成c-jun和NFκB轉錄的活化；另外，亦包括抑癌基因p53受到抑制或突變。然而，目前在診斷上仍然還沒有一個很好的標誌，可以用來判斷肝癌；因此，透過肝癌區與鄰近非肝癌區基因表現的差異，藉而用來尋找肝癌標誌；並可藉由分組比較來了解不同癌期、男女及不同區之間的關係。透過RT-PCR的方式，針對IGF-I、IGF-II、ER-α、PPAR-α、PPARγ、β-catenin及HBx作定量，並經由分類後，對於肝癌 (Hepatocellular carcinoma, HCC) 區與鄰近非肝癌 (nontumor liver) 區mRNA表現的比較，發現PPAR-γ,β-catenin,及 PPAR-α mRNA的37個病人中，其肝細胞癌組織的mRNA量要比肝細胞癌鄰近正常組織來得高，且前二者有統計上的極顯著差異 (P<0.01)；後者在統計上含有顯著差異 (P<0.05)。在IGF-I及IGF-II mRNA，在帶有HBV的肝癌患者，其肝細胞癌鄰近正常組織要比肝細胞癌組織來的高，且分別有極顯著及顯著差異；相反地，PPAR-α及PPAR-γ及β-catenin在癌區中的mRNA含量遠大於鄰近正常組織，且PPAR-α及PPAR-γ和HCV型肝癌極相關(P<0.01) 。在分期上，PPAR-γ的變化多發生在第一期HCC而可能和早期肝癌的發生有關，IGF-I及IGF-II變化多發生於第三期HCC病人，可能和中期肝癌有關；β-catenin則變化多發生於第四期HCC病人，和末期肝癌可能有關。另外，ER-α則在肝細胞癌鄰近正常組織中表現較多，而肝細胞癌組織中，表現少，幾乎不可測。由以上對IGF-I、IGF-II、ER-α、PPAR-α、PPARγ、β-catenin及HBx mRNA主要表現處做相關分析，可間接了解男女、期數及不同區間與HBV、HCV等因子與肝癌行成的相關性；這或許亦啟開肝癌研究的另一扇門。
I.
According to statistics epidemiological and experimental studies, the incidence of hepatocellular carcinoma (HCC) is higher in men than in women. In addition to the varied life style, the regulation of different sex hormone is probably the major cause, although the exact mechanism remains to be determined. Besides, it was found that estrogen can to suppress hepatogenesis in animal model. Therefore, in our study, Hep3B cells were administrated estrogen and estrogen-like compounds to identify that apoptosis could be induced. Firstly, after treatments of 10-6 and 10-8M estrogen, and estrogen-like compounds, Hep3B cells showed the occurrence of apoptosis by DNA fragmentation, DAPI and cytochrome -c expression (by western blotting), declined survival potential by MTT assay, and decreased drug-resistance by GST activity assay. Secondly, after transfection of WtER-α and DnER-α, and treatments with estrogen, Hep3B cells demonstrated the reduction of STAT 3 that is a proliferation- enhancing transcription factor. It was also observed that suppressed proliferation by MTT assay after treatment of 10-6M estrogen,Tamoxifen and Genistein, and reduction of GST activity in 10-6M estrogen and Genistein. Taken together, estrogen and estrogen-like compounds can induce apoptosis in Hep3B cells, and among them, estrogen,Tamoxifen and Genistein showed the strongest potential. Furthermore, inhibition of proliferation can be promoted by WtER-α in Hep3B cells as well. Therefore, the combination of estrogen treatment and enhanced-ER would provide further benefit on inhibition of proliferation in hepatoma cells.
II.
The occurrence of HCC could be resulted from several factors associated with genetic alterations, such as the activation of oncogenes, the enhancement of c-jun and NFkB caused by HBV, the inhibition or mutation of antitumor gene,p53. However, in the present, there has not been good marker to diagnose HCC yet. Therefore, the analysis of gene expression in tumor and non-tumor tissue, become necessary to find the marker of the development of HCC. In the study, from the result of RT-PCR analysis focused on IGF-I, IGF-II, ER-α, PPAR-α, PPAR-γ, β-catenin and HBx from tumor and non-tumor tissue, it was found that the mRNA levels of PPAR-α, PPAR-γ, β-catenin are significantly higher in tumor areas than in non-tumor area. Particularly, PPAR-α, PPAR-γ have very close correlation in patients with HCV at P<0.05 and 0.01. Conversely, in HBV patients, the mRNA level of IGF-I and IGF-II were significantly lower in tumor area than in non-tumor area. In addition, the elevation of PPAR-γ mRNA level occurred at the first stage, indicating the possible relation with early stage of HCC. The alteration of IGF-I/IGF-II, and β-catenin mRNA levels appeared at the third and fourth stage, respectively, suggesting the possible correlation with middle and final stage, respectively, of HCC. Furthermore, gene expression of ER-α was more in non-tumor tissue than in tumor tissue. Taken together, based on the divisions of sex, stage, area of tumor tissue of HCC, and factors of HBV/HCV, the correlation between mRNA of IGF-I, IGF-II ER-α, PPAR-α, PPAR-γ, β-catenin and the occurrence of HCC would provide a different view for the future research of HCC. |
