本文選題：豬肝細(xì)胞 + 原代肝細(xì)胞��； 參考：《華中科技大學(xué)》2010年博士論文

【摘要】： 第一部分：前期實(shí)驗(yàn)——豬原代肝細(xì)胞的分離、培養(yǎng)及可逆性永生化 [目的]建立大動(dòng)物肝組織灌流分離原代肝細(xì)胞的方法；建立用攜帶永生化基因的逆轉(zhuǎn)錄病毒感染原代肝細(xì)胞實(shí)現(xiàn)肝細(xì)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(xì)胞,然后用SSR#69包裝細(xì)胞釋放的攜帶Cre/LoxP系統(tǒng)／SV40T及潮霉素抗性基因的逆轉(zhuǎn)錄病毒感染豬原代肝細(xì)胞,潮霉素篩選陽(yáng)性克隆細(xì)胞團(tuán),克隆環(huán)挑選目的克隆增生細(xì)胞團(tuán)進(jìn)行單獨(dú)傳代培養(yǎng)。最后添加Cre重組酶,切除兩個(gè)LoxP位點(diǎn)間的永生化基因SV40T,實(shí)現(xiàn)永生化的逆轉(zhuǎn)。 [結(jié)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(xì)胞(產(chǎn)量：9.4±7.5×106／g肝組織；活性：94.6±4.2%)。在此基礎(chǔ)上利用SSR#69釋放的含有SV40T的逆轉(zhuǎn)錄病毒轉(zhuǎn)染豬肝細(xì)胞獲得成功,永生化的肝細(xì)胞具有正常肝細(xì)胞的形態(tài)特點(diǎn),能傳代培養(yǎng)。加入Cre重組酶逆轉(zhuǎn)永生化后,肝細(xì)胞停止增生。 [結(jié)論]我們建立了大動(dòng)物肝組織四步循環(huán)灌流法分離原代肝細(xì)胞,并成功實(shí)現(xiàn)了用攜帶永生化基因SV40T的逆轉(zhuǎn)錄病毒轉(zhuǎn)染豬原代肝細(xì)胞,獲得了永生化的豬肝細(xì)胞。 第二部分：人原代肝細(xì)胞的分離、培養(yǎng) [背景及目的]藥物代謝實(shí)驗(yàn)、生物人工肝及細(xì)胞移植等均需要大量的人肝細(xì)胞,而目前國(guó)際上利用外科手術(shù)切除后廢棄的健康肝組織分離原代肝細(xì)胞的經(jīng)驗(yàn)尚少。本部分實(shí)驗(yàn)旨在建立一種高效利用手術(shù)切除后廢棄的健康肝組織分離人原代肝細(xì)胞的方法。 [方法]收集良性肝病患者外科手術(shù)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(xì)胞。同時(shí)評(píng)價(jià)獲取肝組織的重量、冷熱缺血時(shí)間等對(duì)肝細(xì)胞分離效果的影響。 [結(jié)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(nèi)膽管結(jié)石患者3例。所有患者查HCV-Ab、HIV-Ab及HBV-Ag均為陰性�；颊呤中g(shù)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(shí)間(WIT) 10-35min,、所有13例組織塊的冷缺血時(shí)間(CIT)均35 min。分離獲得的細(xì)胞產(chǎn)量為4.8±2.1×106／g肝組織,臺(tái)酚藍(lán)染色后用血球計(jì)數(shù)板測(cè)肝細(xì)胞活率為78.1±10.4%(62%-93%)。 [結(jié)論]1,良性肝病(主要是肝血管瘤及肝內(nèi)膽管結(jié)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(xì)胞。2,對(duì)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(xì)胞分離效果。3,應(yīng)盡量縮短獲取肝組織的熱缺血時(shí)間,提高肝細(xì)胞分離效果。4,對(duì)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(xì)胞效果欠佳。 第三部分：人肝細(xì)胞的可逆性永生化 [背景及目的]人原代肝細(xì)胞體外培養(yǎng)時(shí)增殖困難,且存在培養(yǎng)時(shí)間短,不能及時(shí)獲得等缺陷,使其應(yīng)用受到很大的限制。具有正常肝細(xì)胞功能的永生化人源肝細(xì)胞,能夠解決細(xì)胞來(lái)源緊缺的難題。本部分實(shí)驗(yàn)旨在利用逆轉(zhuǎn)錄病毒實(shí)現(xiàn)SV40T單個(gè)永生化基因的轉(zhuǎn)染,構(gòu)建人源肝細(xì)胞的可逆性永生化。 [方法]用SSR#69包裝細(xì)胞釋放的攜帶Cre/LoxP系統(tǒng)／SV40T及潮霉素抗性基因的逆轉(zhuǎn)錄病毒轉(zhuǎn)染人原代肝細(xì)胞,7天后用潮霉素篩選陽(yáng)性克隆細(xì)胞團(tuán),用合適大小的克隆環(huán)挑選目的克隆增生的細(xì)胞團(tuán)進(jìn)行單獨(dú)傳代培養(yǎng)。最后添加Cre重組酶,以切除兩個(gè)LoxP位點(diǎn)間的永生化基因SV40T,實(shí)現(xiàn)永生化的逆轉(zhuǎn)。RT-PCR及免疫熒光染色驗(yàn)證永生化基因SV40T的存在。 [結(jié)果]逆轉(zhuǎn)錄病毒轉(zhuǎn)染7天,潮霉素進(jìn)行篩選14天后,培養(yǎng)瓶中出現(xiàn)大量克隆增生的細(xì)胞團(tuán),形態(tài)類似肝細(xì)胞�？寺…h(huán)挑選至新培養(yǎng)瓶中單獨(dú)培養(yǎng),細(xì)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(zhuǎn)永生化后,細(xì)胞即停止生長(zhǎng)。RT-PCR檢測(cè)永生化細(xì)胞具有人原代肝細(xì)胞的標(biāo)志基因,免疫熒光染色亦驗(yàn)證永生化細(xì)胞內(nèi)SV40T的存在。 [結(jié)論]我們建立的可逆性永生化人源肝細(xì)胞,具有肝細(xì)胞的功能,體外能傳代培養(yǎng)。更重要的是,我們成功實(shí)現(xiàn)了逆轉(zhuǎn)錄病毒單永生化基因轉(zhuǎn)染人原代肝細(xì)胞的方法,為后繼實(shí)驗(yàn)進(jìn)行人肝細(xì)胞SV40T/hTERT雙永生化基因轉(zhuǎn)染,實(shí)現(xiàn)人源肝細(xì)胞的徹底永生化奠定方法學(xué)基礎(chǔ)。
[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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)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(hào)：1954857