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研究生:林玨羽
研究生(外文):Chueh-Yu Lin
論文名稱:HBy基因對MSG2肝細胞轉型之影響
論文名稱(外文):Affects of HBy gene on transformation of MSG2 liver cancer cells
指導教授:曾英傑曾英傑引用關係
指導教授(外文):Yin-Jeh Tzeng
學位類別:碩士
校院名稱:慈濟大學
系所名稱:分子生物及細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:95
語文別:英文
論文頁數:51
中文關鍵詞:肝癌肝癌細胞MSG2HBy基因B型肝炎病毒
外文關鍵詞:Hepatocellular carcinomaHBVHBy geneliver cancer cells MSG2
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肝癌(Hepatocellular carcinoma)是一種世界性的疾病,據世界衛生組織統計,全球每年新發肝癌病例計100萬至150萬例。台灣行政院衛生署公布,肝癌的發生率在男性是每十萬人有二十五人,在女性是是每十萬人有十人。在世界上,有百分之70%以上肝癌發生與B型肝炎病毒有關。在台灣,男性肝癌經B型肝炎病毒感染轉變為肝癌的機率有67%,女性有55.2%。研究認為,B型肝炎病毒 (HBV) 感染後, HBV中的致癌基因HBx被活化所引起的細胞轉化,是引起肝癌的原因之ㄧ。雖然許多研究認為HBV的一個基因HBx對肝炎產生有關,但對於HBV如何造成肝癌的機制仍未有定論。1990年代,Miller等人.提出,在HBx反義股有一個基因的表現框架,可惜到現在仍未有研究報告指出,那段未知基因在癌瘤生物學上的意義有那些。目前我們的實驗室稱那段對側基因為HBy ,並進一步探討它對肝癌細胞的影響。
在本研究中,首先探討HBy是否在攜帶HBV的肝癌細胞株表現。結果顯示HBy mRNA之表現可在MSG2與hepG2.2.15的細胞株得到證實。然而其蛋白質卻未能測得。接著,以MSG2建立攜帶HBy基因的肝癌細胞株,用來探討細胞過度表達外來HBy時,會產生那些生物學上的影響。研究結果發現,當MSG2 表達外來的HBy時,可造成細胞生長速度變快。這項研究結果也在SCID的小鼠中,以皮下注射表達HBy的MSG2細胞之方式,經觀察後得到證實。進一步,分別以胞落形成法 (Colony formation),以及軟洋菜膠細胞集落形成法(soft agar),分析細胞轉型的特性。結果顯示HBy可促使較多之MSG2細胞株在兩種測試法中形成細胞集落。在藥物的敏感實驗中,我們利用苦瓜的萃取素及cisplatin兩種藥物測試HBy 是否影響細胞對藥物之敏感性。經過加入不同的劑量於細胞並隨著時間點的不同來觀察後,發現過度表現HBy的細胞,對苦瓜的耐受性比沒有HBy表現的細胞來得強;但在cisplatin的藥劑試驗中發現HBy表現與否,並不會造成細胞對藥物敏感性的影響。以上結果顯示,HBy具有惡化細胞癌化的效果,這意味著HBy如在感染HBV肝細胞過度表現,可能促進癌化的過程。
接下來的研究,將進一步了解到底HBy表達的時候,細胞有那些基因也跟著被表達出來,而細胞表面那些分子也會跟著被改變表達,如果可以的話,也想進一步了解這些分子的表現和作用是如何被調控。
Hepatocellulax carcinoma (HCC) is one of the human neoplasms associated with viral infections. Based on the statistics of World Health Organization (WHO), 1000,000~1500,000 HCC cases were detected in every year. Department of Health, Executive Yuan of Taiwan reported that there are 25 people in each 1,000,000 males and 10 people in each 1, 000,000 females are the HCC carrier. Hepatitis B virus (HBV) is the most important etiological factors of HCC, accounting for more than 70% of cases worldwide. In Taiwan 67% of males and 55.2% of females HCC are related to HBV infection. Research showed that the activation of HBx gene could transform the normal liver into HCC. Although the relationship between HBx and HCC is well studied, but the mechanism of how HBV leading to HCC is not yet clear.
An open reading frame (ORF6) was first reported in 1990 and locates on the opposite side of HBx coding strand (16). This gene was named HBy by us. The biological implication of this gene (HBy) was not further studied sine then.
In this study, we first examined the HBy expression in liver cancer cell lines containing HBV genome. The HBy mRNA, instead of the HBy protein expression was detected in MSG2 and hepG2.2.15 cell line. To characterize the tumor biological effects of HBy gene, HBy-EGFP fusion gene under the contral of CMV promoter was transfected into the liver cancer cells (MSG2) and overexpressed. The biological effects of HBy in the cells were measured. Our results showed that the CMV-HBy-EGFP transfected cell lines (y-13, y-20 and y-30) grew much faster than mock-transfected ones. This phenomenon was further confirmed in vivo by subcutaneously inoculating the HBy((+)-EGFP(+) as well as parental MSG2 cells into SCID mice and comparing their tumorigenesis potentials. Furthermore, CMV-HBy-GFP transfected cells displayed significantly higher anchorage independent capability than that of mock-transfected cells. In the drug resistant tests, we found that HBy reduced sensitivity of MSG2 cells to balsam pear extracts; whereas HBy did not significantly influence sensitivity of MSG2 cells to cisplatin. Based on these results, we concluded that HBy expression plays an enhancing role in liver tumorigenesis.
Up- or down-regulated genes associated with HBy will be further characterized. Moreover the molecules on cell membrane will be studied on their influences caused by HBy stimulation. Finally, the mechanism of genes affected by HBy induction will be explored.
1. Introduction………………………. …………………………………8
1.1. Hepatocellular carcinoma (HCC) epidemiology………………………8
1.2. Hepatitis B viruses………………………. ……………………………8
1.2.1. The role of HBV related Hepatocellular carcinoma (HCC) …….……8
1.2.2. Structure of HBV genome and a putative gene, HBy…………………9
1.2.3. HBy gene and HBy protein……………………………………………9
1.3. Anti-tumors drugs……………………………………………………10
1.3.1. Effects of balsam pear on liver cancer cells………………………10
1.3.2. Effects of cisplatin on cancer cells………………………………11
1.4. The purpose and experimental design of this thesis………………11
2. Materials and methods………………………………………………12
2.1. Materials………………………………………………………………12
2.1.1. Animals………………………………………………………………12
2.1.2. Plasmid………………………………………………………………12
2.1.3 Cells……………………………………………………………………12
2.1.4. RNA Analysis…………………………………………………………13
2.1.4.1. RNA extraction from the liver cancer cells…………………………13
2.1.4.2. RT-PCR analysis……………………………………………………13
2.1.5. Protein Analysis………………………………………………………13
2.1.5.1. Protein extraction from the liver cancer cell………………………13
2.1.5.2. Western blot analysis………………………………………………14
2.1.6. Immunofluorescence staining……………………………………15
2.1.7. Colony formation assay………………………………………………15
2.1.8. Anchorage-independent growth assay in soft agar……………………16
2.1.9. WST-1 assay…………………………………………………………16
2.2. Methods………………………………………………………………17
2.2.1. Cell transfection………………………………………………………17
2.2.2. RNA Analysis…………………………………………………………17
2.2.2.1. RNA was extracted from the diffetent liver cancer cells……………17
2.2.2.2. RT-PCR analysis………………………………………………18
2.2.3. Protein Analysis……………………………………………………19
2.2.3.1. Protein extraction from the different liver cancer cells………………19
2.2.3.2 Western blot analysis…………………………………………………19
2.2.4. Immunofluorescence staining……………………………………20
2.2.5. Cell proliferation assay…………………………………………20
2.2.6. Colony formation assay…………………………………………21
2.2.7. Anchorage-independent growth assay in soft agar……………21
2.2.8. Tumorigenicity assay inSCID mouse………………………………22
2.2.9. Effects of HBy on sensitivity of MSG2 cells to anti-tumor drugs………22
2.2.9.1. To detect the effect of HBy on balsam pear sensitivity of MSG2 cells…22
2.2.9.2. Effect of HBy on cisplatin sensitivity of MSG2 cells……………………22
2.2.10. To detect the effects of HBy on cell adhesion of MSG2 cells……………23
3. Results………………………………24
3.1. In vitro identification of HBy expression in HBV positive cancer cells……24
3.2. Generation HBy of MSG2 liver cancer cells overexpressing HBy…………24
3.2.1. MSG2 liver cancer cells transfected with CMV-HBy-EGFP gene………24
3.2.2. Expression of HBy-EGFP mRNA in MSG2 cells………………………25
3.2.3. Expression of HBy-EGFP protein in MSG2 cells………………………25
3.2.4. HBy-EGFP is localized in nucleus and cytoplasm of MSG2 cells………26
3.3. Effects of HBy on cell growth………………………………………………27
3.3.1. HBy induces proliferation of MSG2 cells and cos7 cells…………………27
3.3.2. HBy induces tumor growth potential in SCID mice………………………27
3.3.3. HBy gene increases colony formation of MSG2 cells………………………28
3.3.4. HBy gene promotes anchorage-independent growth potential of MSG2 cells in soft agar………………28
3.4. Effects of HBy on sensitivity of MSG2 cells to anti-tumor drugs…………29
3.4.1. HBy reduces sensitivity of MSG2 cells to balsam pear extracts…………29
3.4.2 HBy does not significantly influence sensitivity ofMSG2 cells to cisplatin……29
3.5. HBy reduces tempory adhesion of MSG2 cells to culture dishes…………30
4. Discussion…………………………………………………………………31
5. References…………………………………………………………………33

List of figures

Fig.1. Map of HBV form I DNA in the cell nucleus.……..…………..............37
Fig.2. The effect of the CMV-HBY(+)-EGFP(+) vector on the MSG2 cells…38
Fig.3. RT-PCR electrophoregram shows HBy expression in HBV(+) and CMV- HBy(+)-EGFP(+) cells ……39
Fig.4. HBy-EGFP fusion protein was expression in MSG2 cell by using western blot, whereas HBy protein was not dectectable in HBV(+) cells…...……40
Fig.5. HBy-EGFP is localized in nucleus and cytoplasm by using confocal immunofluorescence microscopy………..…………………………. ……41
Fig.6. HBy enhances proliferation of MSG2 cells……………….……....….42
Fig.7. HBy gene overexpression increases tumorigenicity of MSG2 cells in SCID mice....…43
Fig.8. HBy expression increase colony formation potential in MSG2 cells… 44
Fig.9. HBy expression increase colony formation potential of MSG2 cells in soft agar….....45
Fig.10. Different levels of resistance to treatments of balsam pear extracts were observed in HBy(+)-GFP(+)/MSG2 cell lines………………………………46
Fig.11. Sensitivity of HBy(+)-EGFP(+),HBy(+)-EGFP(+) and parental MSG2 cells to cisplatin treatment in a dose response manner…………………………….48
Fig.12. HBy reduces temporal adhesion of MSG2 cells to culture dishes……49
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