稳定表达TNF-α及其突变体基因的人源细胞模型的建立
发布时间：2018-08-31 08:13
【摘要】： 肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)是一种重要的炎症因子,主要由激活的单核/巨噬细胞产生。TNF-α在机体的免疫应答与炎症反应中起着重要作用,控制着细胞分化、增殖与凋亡。它以两种形式存在:26kDa的膜型TNF-α(mTNF-α)与17kDa的可溶型TNF-α(sTNF-α),sTNF-α由mTNF-α酶解而来。两型TNF-α通过与两型受体(TNFR1与TNFR2)结合而发挥广泛的生物学作用,但两型TNF-α的胞毒作用方式和机制不尽相同。此外,mTNF-α除了作为配体与TNFR结合后向靶细胞传递正向信号而发挥生物学效应外,还可同时作为受体向效应细胞传递反向信号(reverse signaling)。 本室前期工作已经建立了能分泌含三种TNF-α基因(野生型TNF-α、膜型TNF-α突变体和分泌型TNF-α突变体)的重组逆转录病毒的包装细胞。本研究首先培养这三株包装细胞,分别收集高滴度逆转录病毒,感染自身TNF-α表达量极低的人骨肉瘤细胞系(MG63),用G418筛选两轮得到稳定表达wTNF-α、mTNF-α和sTNF-α蛋白的细胞模型,为进一步研究两型TNF-α的生物学功能以及信号转导通路等提供实验工具。 一、鉴定MG63细胞TNF-α的表达 同等条件下分别提取MG63细胞和HL-60细胞的总mRNA进行RT-PCR检测,经过35个循环后,取相同量的产物进行电泳,结果证实:MG63细胞中TNF-αmRNA水平极低,明显低于HL-60细胞。同时,在相同条件下分别提取MG63细胞和HL-60细胞的总蛋白进行Western blotting检测,结果显示HL-60细胞可见明显的TNF-α条带,MG63细胞仅出现一条微弱的条带,而两者的β-actin蛋白量一致,提示MG63细胞的TNF-α表达量很低。此外,流式细胞术检测结果显示:MG63细胞的TNF-α表达量为0.53%;ELISA检测其培养上清中表达的sTNF-α低于检测水平。 二、稳定表达TNF-α及其突变体细胞模型的建立 复苏本室前期工作建立的表达TNF-α及其突变体基因的重组逆转录病毒的包装细胞克隆,收集高滴度逆转录病毒,感染MG63细胞,经过筛选建立了表达TNF-α及其突变体基因的三种细胞模型。用各种方法鉴定证实TNF-α及其突变体基因在MG63细胞中得到有效表达。FCM结果显示:MG63/wTNF细胞表达TNF-α量达29.97%;MG63/mTNF细胞表达TNF-α的量高达70.84%;然而MG63/sTNF细胞表面也有少量的TNF-α的表达(9.15%),可能是由于细胞表面的TNFR结合了部分培养上清中的sTNF-α所致。ELISA检测证实MG63/wTNF细胞和MG63/sTNF细胞的培养上清中sTNF-α的含量很高,分别为437.16pg/ml、380.95pg/ml;而MG63/mTNF细胞的培养上清中sTNF-α的表达量低于检测水平。免疫荧光结果显示MG63/sTNF细胞表面有微弱的荧光,呈环状分布;而MG63/wTNF细胞和MG63/mTNF细胞可见有很强的绿色荧光。Western blotting检测的结果表明:MG63/wTNF细胞可见有26kD的mTNF-α和17kD的sTNF-α,但sTNF-α的量远远低于mTNF-α,仅见微弱的条带;MG63/mTNF细胞仅见明显的26kD的mTNF-α的表达;MG63/sTNF细胞仅见微弱的26kD mTNF-α,但其培养上清中可见有明显的17kD sTNF-α的表达。上述结果均提示这三种细胞模型建立成功。 三、TNF-α及其突变体的生物活性 采用TNF-α敏感细胞株L929鉴定TNF-α及其突变体的生物学活性。将三种细胞株培养至对数生长期,取相同细胞数培养24～48h,收集培养细胞和上清分别与靶细胞细胞共孵育,进行TNF-α胞毒活性的检测。结果显示:固定的MG63/mTNF细胞具有胞毒活性(64.28%);MG63/sTNF细胞的培养上清亦具有胞毒活性(30.65%);MG63/wTNF细胞的固定细胞和培养上清均有胞毒活性(37.79%、38.02%)。此外,细胞的胞毒效应与其TNF-α的表达量呈正相关,并且胞毒效应均能被TNF-α单抗所阻断(p0.01),提示其胞毒活性为TNF-α特异性的。 四、细胞表达TNF-α基因的持久性检测 本研究的目的是建立稳定表达目的基因的细胞模型,故为了观察细胞表达目的基因的持久性,采用了FCM定期测定连续培养的细胞模型TNF-α表达量的改变。检测结果显示:在G418维持培养下,细胞株经过6次传代培养约4周左右,TNF-α的表达量从86.23%下降至50%左右;而无G418维持培养的细胞株,经过约5次传代培养约2.5周左右,TNF-α的表达量下降至50%。 此外,由于细胞克隆在长时间传代培养后,目的基因的表达率明显下降。为了维持较高表达目的基因的细胞克隆,将复苏的细胞克隆(FCM检测其TNF-α的表达率为21.29%)再次进行亚克隆筛选,从而获得了目的基因表达较高的亚克隆,FCM检测其mTNF-α的表达率达到87.08%。 结论:成功建立稳定表达野生型、跨膜型和分泌型TNF-α基因的人源细胞模型。三种细胞模型表达的TNF-α及其突变体基因均具有生物学活性。
[Abstract]:Tumor necrosis factor-alpha (TNF-alpha) is an important inflammatory factor, mainly produced by activated monocytes/macrophages. TNF-alpha plays an important role in the body's immune response and inflammatory response, controlling cell differentiation, proliferation and apoptosis. It exists in two forms: 26 kDa membrane TNF-alpha (mTNF-alpha) and 17 kDa. Soluble TNF-alpha (sTNF-alpha) and sTNF-alpha are derived from the enzymatic hydrolysis of mTNF-alpha. Both types of TNF-alpha play a wide range of biological roles by binding to two types of receptors (TNFR1 and TNFR2), but the cytotoxic mechanisms of the two types of TNF-alpha are different. In addition, mTNF-alpha plays a biological role as a ligand that transmits positive signals to target cells after binding to TNFR. Besides, it can also serve as a receptor to transmit reverse signaling to effector cells.
In this study, three recombinant retroviral packaging cells secreting three kinds of TNF-alpha genes (wild-type, membrane-type and secretory-type) were established. Cell lines (MG63) were screened by G418 for two rounds to obtain a cell model stably expressing wTNF-a, mTNF-a and sTNF-a proteins.
First, identify the expression of TNF- alpha in MG63 cells.
The total mRNA of MG63 cells and HL-60 cells were extracted under the same conditions and detected by RT-PCR. After 35 cycles, the same amount of products were obtained for electrophoresis. The results showed that the level of TNF-alpha mRNA in MG63 cells was very low, significantly lower than that in HL-60 cells. In addition, flow cytometry showed that the expression of TNF-a in MG63 cells was 0.53%; ELISA detected the expression of sTN in the supernatant of MG63 cells. F- alpha was lower than the detection level.
Two, stable expression of TNF- alpha and its mutant cell models were established.
Recombinant retroviruses expressing TNF-alpha and its mutant genes were cloned from the packaged cells of MG63 cells. High titer retroviruses were collected to infect MG63 cells. Three cell models expressing TNF-alpha and its mutant genes were established by screening. The TNF-alpha and its mutant genes were identified by various methods. The results of FCM showed that the expression of TNF-a was 29.97% in MG63/wTNF cells, 70.84% in MG63/mTNF cells, and 9.15% in MG63/sTNF cells, possibly due to the combination of TNFR on the surface of MG63/sTNF cells with sTNF-a in the supernatant of some cultures. The content of sTNF-a in the supernatant of TNF cells and MG63/sTNF cells was very high, 437.16 pg/ml and 380.95 pg/ml, respectively, while the expression of sTNF-a in the supernatant of MG63/mTNF cells was lower than the detected level. The results of Western blotting showed that MG63/wTNF cells had 26 kD mTNF-a and 17 kD sTNF-a, but the amount of sTNF-a was much lower than that of mTNF-a, and only slight bands were observed; MG63/mTNF cells had only 26 kD mTNF-a expression; MG63/sTNF cells had only slight 26 kD mTNF-a expression, but the supernatant of MG63/sTNF cells was medium. The results showed that the expression of 17kD sTNF- alpha was obvious. All these results indicated that the three cell models were successfully established.
Three, bioactivity of TNF- alpha and its mutants.
TNF-a sensitive cell line L929 was used to identify the biological activities of TNF-a and its mutants. Three kinds of cell lines were cultured at logarithmic growth stage. The same number of cells were cultured for 24-48 hours. The cultured cells and supernatants were collected and co-incubated with the target cells, respectively. The cytotoxicity of TNF-a was detected in the immobilized MG63/mTNF cells. The cytotoxic activity of MG63/sTNF cell culture supernatant (30.65%) and MG63/wTNF cell immobilization and culture supernatant (37.79%, 38.02%) were positive correlated with the expression of TNF-a, and the cytotoxic effect was blocked by TNF-a monoclonal antibody (p0.01). TNF- alpha is specific.
Four, the persistence of cell expression of TNF- alpha gene.
The aim of this study was to establish a cell model with stable expression of the target gene. In order to observe the persistence of the target gene, the expression of TNF-alpha in the cell model was measured periodically by FCM. The expression of TNF-alpha decreased from 86.23% to 50% and the expression of TNF-alpha decreased to 50% after about 2.5 weeks of subculture without G418.
In addition, in order to maintain the high expression of the target gene, the resuscitated cell clones (21.29% of which were detected by FCM) were subcloned and screened again, and the subclones with high expression of the target gene were obtained. The expression rate of mTNF- alpha reached 87.08%.
CONCLUSION: Human cell models with stable expression of wild type, transmembrane type and secretory type TNF-alpha genes were successfully established.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2214415
[Abstract]:Tumor necrosis factor-alpha (TNF-alpha) is an important inflammatory factor, mainly produced by activated monocytes/macrophages. TNF-alpha plays an important role in the body's immune response and inflammatory response, controlling cell differentiation, proliferation and apoptosis. It exists in two forms: 26 kDa membrane TNF-alpha (mTNF-alpha) and 17 kDa. Soluble TNF-alpha (sTNF-alpha) and sTNF-alpha are derived from the enzymatic hydrolysis of mTNF-alpha. Both types of TNF-alpha play a wide range of biological roles by binding to two types of receptors (TNFR1 and TNFR2), but the cytotoxic mechanisms of the two types of TNF-alpha are different. In addition, mTNF-alpha plays a biological role as a ligand that transmits positive signals to target cells after binding to TNFR. Besides, it can also serve as a receptor to transmit reverse signaling to effector cells.
In this study, three recombinant retroviral packaging cells secreting three kinds of TNF-alpha genes (wild-type, membrane-type and secretory-type) were established. Cell lines (MG63) were screened by G418 for two rounds to obtain a cell model stably expressing wTNF-a, mTNF-a and sTNF-a proteins.
First, identify the expression of TNF- alpha in MG63 cells.
The total mRNA of MG63 cells and HL-60 cells were extracted under the same conditions and detected by RT-PCR. After 35 cycles, the same amount of products were obtained for electrophoresis. The results showed that the level of TNF-alpha mRNA in MG63 cells was very low, significantly lower than that in HL-60 cells. In addition, flow cytometry showed that the expression of TNF-a in MG63 cells was 0.53%; ELISA detected the expression of sTN in the supernatant of MG63 cells. F- alpha was lower than the detection level.
Two, stable expression of TNF- alpha and its mutant cell models were established.
Recombinant retroviruses expressing TNF-alpha and its mutant genes were cloned from the packaged cells of MG63 cells. High titer retroviruses were collected to infect MG63 cells. Three cell models expressing TNF-alpha and its mutant genes were established by screening. The TNF-alpha and its mutant genes were identified by various methods. The results of FCM showed that the expression of TNF-a was 29.97% in MG63/wTNF cells, 70.84% in MG63/mTNF cells, and 9.15% in MG63/sTNF cells, possibly due to the combination of TNFR on the surface of MG63/sTNF cells with sTNF-a in the supernatant of some cultures. The content of sTNF-a in the supernatant of TNF cells and MG63/sTNF cells was very high, 437.16 pg/ml and 380.95 pg/ml, respectively, while the expression of sTNF-a in the supernatant of MG63/mTNF cells was lower than the detected level. The results of Western blotting showed that MG63/wTNF cells had 26 kD mTNF-a and 17 kD sTNF-a, but the amount of sTNF-a was much lower than that of mTNF-a, and only slight bands were observed; MG63/mTNF cells had only 26 kD mTNF-a expression; MG63/sTNF cells had only slight 26 kD mTNF-a expression, but the supernatant of MG63/sTNF cells was medium. The results showed that the expression of 17kD sTNF- alpha was obvious. All these results indicated that the three cell models were successfully established.
Three, bioactivity of TNF- alpha and its mutants.
TNF-a sensitive cell line L929 was used to identify the biological activities of TNF-a and its mutants. Three kinds of cell lines were cultured at logarithmic growth stage. The same number of cells were cultured for 24-48 hours. The cultured cells and supernatants were collected and co-incubated with the target cells, respectively. The cytotoxicity of TNF-a was detected in the immobilized MG63/mTNF cells. The cytotoxic activity of MG63/sTNF cell culture supernatant (30.65%) and MG63/wTNF cell immobilization and culture supernatant (37.79%, 38.02%) were positive correlated with the expression of TNF-a, and the cytotoxic effect was blocked by TNF-a monoclonal antibody (p0.01). TNF- alpha is specific.
Four, the persistence of cell expression of TNF- alpha gene.
The aim of this study was to establish a cell model with stable expression of the target gene. In order to observe the persistence of the target gene, the expression of TNF-alpha in the cell model was measured periodically by FCM. The expression of TNF-alpha decreased from 86.23% to 50% and the expression of TNF-alpha decreased to 50% after about 2.5 weeks of subculture without G418.
In addition, in order to maintain the high expression of the target gene, the resuscitated cell clones (21.29% of which were detected by FCM) were subcloned and screened again, and the subclones with high expression of the target gene were obtained. The expression rate of mTNF- alpha reached 87.08%.
CONCLUSION: Human cell models with stable expression of wild type, transmembrane type and secretory type TNF-alpha genes were successfully established.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 李清芬,李卓娅,龚非力,姜晓丹,徐勇,冯玮,熊平;稳定高效表达TNF-α基因及其突变体的H22肿瘤细胞株的建立[J];细胞与分子免疫学杂志;2001年03期
2 石文芳,李卓娅,龚非力,熊平,徐勇;跨膜型与分泌型TNF-α细胞毒效应的比较[J];中华微生物学和免疫学杂志;1998年06期
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