hTERT基因转染建立永生化山羊卵巢间质细胞系的研究
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摘要
卵巢间质细胞的体外培养是研究卵巢功能和作用机理的重要手段,试验中使用细胞系具有很多优点,如细胞纯度高、性质稳定和操作简便等。山羊卵巢间质细胞系的建立可以为卵巢的进一步研究提供良好的试验材料。本试验在原代培养的基础上,将目的质粒pCI-neo-hTERT转入山羊卵巢间质细胞中,进行永生化研究,结果如下:
1.无菌采取山羊卵巢,将组织剪碎后加入适量1.5 mg/mL的Ⅱ型胶原酶消化3h,经过差速贴壁法纯化后于37℃,5%的CO2条件下培养。培养的细胞贴壁生长,呈长梭形或多角形,单层细胞交织成网状。免疫细胞化学鉴定表明,所分离细胞呈波形蛋白阳性和角蛋白阴性。
2.采用脂质体转染法,将pCI-neo-hTERT导入第2代山羊卵巢间质细胞,经过400μg/mL G418筛选,获得阳性克隆,酶消化后传代扩大培养。传代培养过程中卵巢间质细胞形态与原代细胞一致,为成纤维样细胞。细胞在体外长期连续培养下生长良好,至今已传至53代。对转染前后细胞进行hTERT免疫细胞化学染色,转染后第50代细胞呈hTERT阳性,未转染细胞呈hTERT阴性,证明hTERT基因已经稳定地整合到转染细胞的基因组中,并且得到了稳定的表达。转染后山羊卵巢间质细胞免疫细胞化学染色仍表现为波形蛋白阳性,角蛋白阴性,具有转染前细胞的特性,细胞表面标志正常。细胞生长曲线表明转染细胞仍具有接触抑制性,保持了原代细胞的生物学特征。
3.对未转染细胞和转染后传至第50代的细胞分别进行细胞周期和凋亡率的测定,结果显示转染后细胞具有正常的细胞增殖周期,且处于S期的细胞比例明显比未转染细胞比例大,说明转染后处于分裂期的细胞较多,细胞增殖旺盛。第50代细胞的凋亡率明显低于原代细胞,活细胞率在90%以上。第50代细胞的倍体分析结果显示,转染后细胞仍具有正常的二倍体。对细胞进行软琼脂克隆试验,发现转染前后细胞均不能在软琼脂中生长,说明细胞无致瘤性,仍为正常细胞,无转化特征。
综上所述,本试验在分离培养山羊卵巢间质细胞的基础上,将人端粒酶逆转录酶(hTERT)基因成功导入培养的原代细胞中,激活了细胞体内的端粒酶活性,延长了细胞的寿命,建立了永生化山羊卵巢间质细胞系。转染细胞系无致瘤性转化,同时保持了正常细胞的生物学特性,为进一步研究奠定了基础。
The culture in vitro of ovarian interstitial cells(OICs) is an important method to study the ovarian functions and action mechanism, there are many advantages to use the cell lines in the experiments, such as the higy purity, the stable characters and the user-friendly control. The establishment of caprine OIC line will offer excellent experimental materials for the further study about ovary. In this study, the aim plasmid namely pCI-neo-hTERT was transfected to primary caprine ovarian interstitial cells (COICs) to be immortalized. Experimental results obtained as follows:
1. Caprine ovaries were collected on the aseptic condition and then cutted, incubated for 3 hours with proper amount collagenase II ( the concentration is 1.5mg/mL), purified with differential velocity adherent technique , cultured at 37℃and 5% CO2. The result showed that the culture cells adhered to the wall, presented long fusiform or mutiple shape, a confluent monolayer was former with the cells being interlaced network. In the immunocytochemical experiment, the isolated cells were positive vimentin and negative keratin.
2. The second generation caprine ovarian interstitial cells (COICs) were transfected with pCI-neo-hTERT by lipofectamine. Cells were screened with the optimum screening concentration, which was 400μg/ml G418, for 14 days. positive clones was acquired and then digested to enlarged passage culture. The transfected COICs remain consistent in the progress of passage culture, namely fibroblast-like as same as primary cells. Cells grow well until the 53rd generation now. In the immunocytochemical staining, the 50th generation cells transfected by hTERT showed positive of hTERT while the untransfected cells were negative, which proved that hTERT gene has integrated in the cellular genome and at the same time expressed stably. The transfected cells still showed positive vimentin and negative keratin with the normal surface marker. Cells transfected by hTERT has contact inhibition like the primary cells according to the cell curves, which shows the ordinary biology characteristics of the immortalized cells.
3. The cell cycle analysis of the untransfected second generation cells and transfected 50th generation cells demonstrated that the immortalized cells has the normal cell cycle and the occupation probabilityof s-phase is obvious large than untransfected cells. The apoptosis results show that apoptosis rate of transfected cells is distinct higher and the living cells dominated over 90%. The DNA ploidy analysis results illustrate that the transfected 50th generation cells remain normal control diploid. In the soft agar cloneing test of untransfected and transfected cells as well as a positive control namely SP20, The COICs including untransfected and transfected cells could not grow in the soft agar, which shows there were no neoplastic transformation in thest immortalized cells.
In summary, hTERT gene was transfected into the caprine ovarian interstitial cells with the basic of the primary cells successfully isolated and cultured in this study. Telomerase was activated in the cells transfected by hTERT gene, which extended the cellular lifespan enormously, so the immortalized caprine ovarian interstitial cell line was established. The transfected cell line shows no neoplastic transformation and at the same time maintains normal cells’biological property, which is a foundation for the further studies.
1.无菌采取山羊卵巢,将组织剪碎后加入适量1.5 mg/mL的Ⅱ型胶原酶消化3h,经过差速贴壁法纯化后于37℃,5%的CO2条件下培养。培养的细胞贴壁生长,呈长梭形或多角形,单层细胞交织成网状。免疫细胞化学鉴定表明,所分离细胞呈波形蛋白阳性和角蛋白阴性。
2.采用脂质体转染法,将pCI-neo-hTERT导入第2代山羊卵巢间质细胞,经过400μg/mL G418筛选,获得阳性克隆,酶消化后传代扩大培养。传代培养过程中卵巢间质细胞形态与原代细胞一致,为成纤维样细胞。细胞在体外长期连续培养下生长良好,至今已传至53代。对转染前后细胞进行hTERT免疫细胞化学染色,转染后第50代细胞呈hTERT阳性,未转染细胞呈hTERT阴性,证明hTERT基因已经稳定地整合到转染细胞的基因组中,并且得到了稳定的表达。转染后山羊卵巢间质细胞免疫细胞化学染色仍表现为波形蛋白阳性,角蛋白阴性,具有转染前细胞的特性,细胞表面标志正常。细胞生长曲线表明转染细胞仍具有接触抑制性,保持了原代细胞的生物学特征。
3.对未转染细胞和转染后传至第50代的细胞分别进行细胞周期和凋亡率的测定,结果显示转染后细胞具有正常的细胞增殖周期,且处于S期的细胞比例明显比未转染细胞比例大,说明转染后处于分裂期的细胞较多,细胞增殖旺盛。第50代细胞的凋亡率明显低于原代细胞,活细胞率在90%以上。第50代细胞的倍体分析结果显示,转染后细胞仍具有正常的二倍体。对细胞进行软琼脂克隆试验,发现转染前后细胞均不能在软琼脂中生长,说明细胞无致瘤性,仍为正常细胞,无转化特征。
综上所述,本试验在分离培养山羊卵巢间质细胞的基础上,将人端粒酶逆转录酶(hTERT)基因成功导入培养的原代细胞中,激活了细胞体内的端粒酶活性,延长了细胞的寿命,建立了永生化山羊卵巢间质细胞系。转染细胞系无致瘤性转化,同时保持了正常细胞的生物学特性,为进一步研究奠定了基础。
The culture in vitro of ovarian interstitial cells(OICs) is an important method to study the ovarian functions and action mechanism, there are many advantages to use the cell lines in the experiments, such as the higy purity, the stable characters and the user-friendly control. The establishment of caprine OIC line will offer excellent experimental materials for the further study about ovary. In this study, the aim plasmid namely pCI-neo-hTERT was transfected to primary caprine ovarian interstitial cells (COICs) to be immortalized. Experimental results obtained as follows:
1. Caprine ovaries were collected on the aseptic condition and then cutted, incubated for 3 hours with proper amount collagenase II ( the concentration is 1.5mg/mL), purified with differential velocity adherent technique , cultured at 37℃and 5% CO2. The result showed that the culture cells adhered to the wall, presented long fusiform or mutiple shape, a confluent monolayer was former with the cells being interlaced network. In the immunocytochemical experiment, the isolated cells were positive vimentin and negative keratin.
2. The second generation caprine ovarian interstitial cells (COICs) were transfected with pCI-neo-hTERT by lipofectamine. Cells were screened with the optimum screening concentration, which was 400μg/ml G418, for 14 days. positive clones was acquired and then digested to enlarged passage culture. The transfected COICs remain consistent in the progress of passage culture, namely fibroblast-like as same as primary cells. Cells grow well until the 53rd generation now. In the immunocytochemical staining, the 50th generation cells transfected by hTERT showed positive of hTERT while the untransfected cells were negative, which proved that hTERT gene has integrated in the cellular genome and at the same time expressed stably. The transfected cells still showed positive vimentin and negative keratin with the normal surface marker. Cells transfected by hTERT has contact inhibition like the primary cells according to the cell curves, which shows the ordinary biology characteristics of the immortalized cells.
3. The cell cycle analysis of the untransfected second generation cells and transfected 50th generation cells demonstrated that the immortalized cells has the normal cell cycle and the occupation probabilityof s-phase is obvious large than untransfected cells. The apoptosis results show that apoptosis rate of transfected cells is distinct higher and the living cells dominated over 90%. The DNA ploidy analysis results illustrate that the transfected 50th generation cells remain normal control diploid. In the soft agar cloneing test of untransfected and transfected cells as well as a positive control namely SP20, The COICs including untransfected and transfected cells could not grow in the soft agar, which shows there were no neoplastic transformation in thest immortalized cells.
In summary, hTERT gene was transfected into the caprine ovarian interstitial cells with the basic of the primary cells successfully isolated and cultured in this study. Telomerase was activated in the cells transfected by hTERT gene, which extended the cellular lifespan enormously, so the immortalized caprine ovarian interstitial cell line was established. The transfected cell line shows no neoplastic transformation and at the same time maintains normal cells’biological property, which is a foundation for the further studies.
引文
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