本文選題：豬肝細胞 + 原代肝細胞　； 參考：《華中科技大學》2010年博士論文

【摘要】： 第一部分：前期實驗——豬原代肝細胞的分離、培養(yǎng)及可逆性永生化 [目的]建立大動物肝組織灌流分離原代肝細胞的方法；建立用攜帶永生化基因的逆轉錄病毒感染原代肝細胞實現(xiàn)肝細胞可逆性永生化的方法。 [方法]采用改良的四步循環(huán)灌流法分離乳豬肝細胞,然后用SSR#69包裝細胞釋放的攜帶Cre/LoxP系統(tǒng)／SV40T及潮霉素抗性基因的逆轉錄病毒感染豬原代肝細胞,潮霉素篩選陽性克隆細胞團,克隆環(huán)挑選目的克隆增生細胞團進行單獨傳代培養(yǎng)。最后添加Cre重組酶,切除兩個LoxP位點間的永生化基因SV40T,實現(xiàn)永生化的逆轉。 [結果]我們共獲得6塊大小合適的豬肝組織,成功分離獲得活性很高的大量原代豬肝細胞(產(chǎn)量：9.4±7.5×106／g肝組織；活性：94.6±4.2%)。在此基礎上利用SSR#69釋放的含有SV40T的逆轉錄病毒轉染豬肝細胞獲得成功,永生化的肝細胞具有正常肝細胞的形態(tài)特點,能傳代培養(yǎng)。加入Cre重組酶逆轉永生化后,肝細胞停止增生。 [結論]我們建立了大動物肝組織四步循環(huán)灌流法分離原代肝細胞,并成功實現(xiàn)了用攜帶永生化基因SV40T的逆轉錄病毒轉染豬原代肝細胞,獲得了永生化的豬肝細胞。 第二部分：人原代肝細胞的分離、培養(yǎng) [背景及目的]藥物代謝實驗、生物人工肝及細胞移植等均需要大量的人肝細胞,而目前國際上利用外科手術切除后廢棄的健康肝組織分離原代肝細胞的經(jīng)驗尚少。本部分實驗旨在建立一種高效利用手術切除后廢棄的健康肝組織分離人原代肝細胞的方法。 [方法]收集良性肝病患者外科手術切除后廢棄的健康肝組織,采用我們自己建立的改良四步循環(huán)灌流法分離人原代肝細胞。同時評價獲取肝組織的重量、冷熱缺血時間等對肝細胞分離效果的影響。 [結果]我們共獲得13塊合適的健康肝組織,其中肝血管瘤患者10例,肝內膽管結石患者3例。所有患者查HCV-Ab、HIV-Ab及HBV-Ag均為陰性。患者手術前肝功能(ALT, ALB等)均在正常水平,平均年齡47歲(23—57歲),血型：O+型4例,A+型6例,AB+型1例,B+型2例。肝組織塊重量為27.2±7.8g(15—42g)。熱缺血時間(WIT) 10-35min,、所有13例組織塊的冷缺血時間(CIT)均35 min。分離獲得的細胞產(chǎn)量為4.8±2.1×106／g肝組織,臺酚藍染色后用血球計數(shù)板測肝細胞活率為78.1±10.4%(62%-93%)。 [結論]1,良性肝病(主要是肝血管瘤及肝內膽管結石)行部分肝切除后廢棄的周邊正常肝組織,可以采用改良的四步循環(huán)灌注法分離獲得大量活性好的人肝細胞。2,對于存在中度以下纖維化的肝組織,如果切緣血管條件好,仍可以有很好的細胞分離效果。3,應盡量縮短獲取肝組織的熱缺血時間,提高肝細胞分離效果。4,對于較小的肝組織塊,切面血管條件欠佳者,采用四步循環(huán)灌流法分離肝細胞效果欠佳。 第三部分：人肝細胞的可逆性永生化 [背景及目的]人原代肝細胞體外培養(yǎng)時增殖困難,且存在培養(yǎng)時間短,不能及時獲得等缺陷,使其應用受到很大的限制。具有正常肝細胞功能的永生化人源肝細胞,能夠解決細胞來源緊缺的難題。本部分實驗旨在利用逆轉錄病毒實現(xiàn)SV40T單個永生化基因的轉染,構建人源肝細胞的可逆性永生化。 [方法]用SSR#69包裝細胞釋放的攜帶Cre/LoxP系統(tǒng)／SV40T及潮霉素抗性基因的逆轉錄病毒轉染人原代肝細胞,7天后用潮霉素篩選陽性克隆細胞團,用合適大小的克隆環(huán)挑選目的克隆增生的細胞團進行單獨傳代培養(yǎng)。最后添加Cre重組酶,以切除兩個LoxP位點間的永生化基因SV40T,實現(xiàn)永生化的逆轉。RT-PCR及免疫熒光染色驗證永生化基因SV40T的存在。 [結果]逆轉錄病毒轉染7天,潮霉素進行篩選14天后,培養(yǎng)瓶中出現(xiàn)大量克隆增生的細胞團,形態(tài)類似肝細胞�？寺…h(huán)挑選至新培養(yǎng)瓶中單獨培養(yǎng),細胞能傳代培養(yǎng)并保持形態(tài)規(guī)則。添加Cre重組酶后逆轉永生化后,細胞即停止生長。RT-PCR檢測永生化細胞具有人原代肝細胞的標志基因,免疫熒光染色亦驗證永生化細胞內SV40T的存在。 [結論]我們建立的可逆性永生化人源肝細胞,具有肝細胞的功能,體外能傳代培養(yǎng)。更重要的是,我們成功實現(xiàn)了逆轉錄病毒單永生化基因轉染人原代肝細胞的方法,為后繼實驗進行人肝細胞SV40T/hTERT雙永生化基因轉染,實現(xiàn)人源肝細胞的徹底永生化奠定方法學基礎。
[Abstract]:Part one: previous experiments - isolation, culture and reversible immortalization of porcine primary hepatocytes
[Objective] to establish a method for isolating primary hepatocytes by perfusion of large animal liver tissue, and to establish a reversible immortalization method of hepatocyte by using retrovirus carrying immortalized gene to infect primary hepatocytes.
[Methods] a modified four step circulatory perfusion method was used to isolate pig hepatocytes, and then the porcine primary hepatocytes were infected with Cre/LoxP system / SV40T and hygromycin resistant retrovirus, and hygromycin was used to screen the positive cloned cell groups. Cloned cloned proliferative cell groups were selected to carry out a single generation culture. Finally, Cre recombinant enzyme was added to remove the immortalized gene SV40T between the two LoxP sites, so as to achieve immortalization reversal.
[results] a total of 6 suitable pig liver tissues were obtained, and a large number of primary porcine liver cells (9.4 + 7.5 * 106 / g liver tissue, 94.6 + 4.2%) were successfully isolated and obtained. On this basis, the transfected porcine hepatocytes, which were released by SSR#69, were successfully transfected into pig hepatocytes, and the immortalized hepatocytes were positive. The morphological characteristics of normal hepatocytes can be subcultured. After adding Cre recombinase to reverse immortalization, the hepatocytes cease to proliferate.
[Conclusion] we established a large animal liver tissue four step circulation perfusion method to separate the primary hepatocytes, and successfully transfected porcine primary hepatocytes with the retrovirus carrying immortalized gene SV40T, and obtained immortalized pig liver cells.
The second part: isolation and culture of human primary hepatocytes
[background and objective] drug metabolism experiments, biological artificial liver and cell transplantation all need a large number of human hepatocytes, but the international experience of separating primary hepatocytes from the abandoned healthy liver tissue after surgical excision is still less. Methods of primary hepatocyte generation.
[Methods] to collect the discarded healthy liver tissues after surgical excision of the patients with benign liver disease, and to separate the human primary hepatocytes by the improved four step circulatory perfusion method established by ourselves. The effects of the weight of liver tissue, the cold and hot ischemic time on the isolation of liver cells were evaluated.
[results] 13 healthy liver tissues were obtained, including 10 cases of hepatic hemangioma and 3 cases of intrahepatic bile duct stones. All patients were examined with HCV-Ab, HIV-Ab and HBV-Ag were negative. The liver function (ALT, ALB, etc.) before operation were at normal level, the average age was 47 years (23 to 57 years), the blood group was 4 cases of O+, 6 cases of A+, 1 cases of AB+, and 2 B+ type. The weight of the liver tissue block was 27.2 + 7.8G (15 - 42g). Hot ischemic time (WIT) 10-35min. The yield of all 13 cases of tissue block cold ischemic time (CIT) 35 min. separation was 4.8 + 2.1 x 106 / g liver tissue, and the rate of liver cell viability was 78.1 + 10.4% (62%-93%) after trypan blue staining.
[conclusion]1, benign liver disease (mainly hepatic hemangioma and intrahepatic bile duct stone) can be separated from the abandoned normal liver tissue after partial hepatectomy. The improved four step perfusion method can be used to separate a large number of active human hepatocytes,.2. For the liver tissue with moderate fibrosis, it can still be good if the marginal vascular conditions are good. The effect of cell separation,.3, should be shortened as far as possible to obtain the thermal ischemia time of the liver tissue, and to improve the effect of liver cell separation,.4. For the smaller liver tissue blocks and the poor cutting conditions, the four step circulation perfusion method is not effective.
The third part: reversible immortalization of human hepatocytes
[background and objective] the proliferation of human primary hepatocytes in vitro is difficult, and there is a short culture time, which can not be obtained in time. The application is greatly restricted. The immortalized human hepatocyte with normal hepatocyte function can solve the problem of cell source shortage. This part of the experiment aims to make use of retrovirus to realize SV40T Transfection of single immortalized gene constructs reversible immortalization of human hepatocytes.
[Methods] human primary hepatocytes were transfected with Cre/LoxP system / SV40T and hygromycin resistance gene released by SSR#69 packaging cells. The positive cloned cell groups were screened by hygromycin 7 days later. The cells were selected by the appropriate size cloned ring to clone the proliferating cell group. Finally, the Cre recombinant enzyme was added to the cell. In addition to the immortalized gene SV40T between the two LoxP loci, the immortalized reverse.RT-PCR and immunofluorescence staining were used to verify the existence of immortalized gene SV40T.
[results] 7 days after retrovirus transfection, a large number of cloned cell clusters appeared in the culture bottle for 14 days. The morphology was similar to hepatocytes. Clones were selected and cultured in the new culture bottle, and the cells could be cultured and kept in shape. After adding Cre recombinant enzyme, the cells stopped growing.RT-PCR detection. Immortalized cells possess the marker genes of human primary hepatocytes, and immunofluorescence staining also confirms the presence of SV40T in immortalized cells.
[Conclusion] the reversible immortalized human hepatocytes have the function of hepatocytes and can be cultured in vitro. More importantly, we have successfully transfected human hepatocytes into human hepatocytes by retrovirus single immortalization gene and transfection of human hepatocyte SV40T/ hTERT double immortalized gene for the successor experiment to realize human hepatocytes. The thorough immortalization lays the foundation of the methodology.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329

【參考文獻】

相關期刊論文 前1條

1 ;Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination[J];World Journal of Gastroenterology;2010年13期

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本文編號：1954857