人抵抗素基因转染对成人脂肪干细胞向成熟脂肪细胞分化的影响
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摘要
抵抗素是一种与胰岛素抵抗和2型糖尿病密切相关的分泌型蛋白,在白色脂肪组织中起重要调节作用,作为新发现的基因之一,其功能尚未完全清楚。抵抗素对啮齿类动物和人类的作用存在一定争议,抵抗素对糖、脂代谢及脂肪组织内的炎症反应一直是人们关注的焦点问题。目前未见文献报道抵抗素过量表达对人脂肪干细胞有何影响及其机制。本研究分离、鉴定成人脂肪干细胞,检测其向成熟脂肪细胞分化过程中不同阶段抵抗素表达,首次应用基因工程重组技术构建携带人类抵抗素基因的慢病毒颗粒Lv-hR并用其感染脂肪干细胞,观察抵抗素转染对脂肪干细胞形态、生长和分化能力的影响。研究抵抗素对脂肪干细胞的作用及机制,阐述抵抗素与肥胖和胰岛素抵抗的关系。研究结果表明：成人脂肪干细胞向成熟脂肪细胞分化过程中抵抗素表达不断下降；抵抗素过表达可使脂肪干细胞分化受抑制,但细胞数量未见明显改变。
Influnce of human resistin transfecting adult adipose-derived stem cell
Adipose tissue was once thought just as a resource of energy, however, it has been seen as an important endocrine tissue with more and more regards recently. It has been proved adipose tissue plays an essential role in regulating human metabolism through its secretion of kinds of protiens such as leptin, adiponectin, resistin, TNF-a ect. Resistin belongs to resistin-like molecules family (RELM), so that is also called FIZZ3. In human and rodents, resistin takes effect in tissues associated with glucose metabolism such as hepatic tissue, muscle and adipose tissue. In particular, administration of recombinant resistin to ob/ob mice, db/db mice and 3T3-L1 preadipocytes in vitro produced insulin resistance, whereas resistin immune neutralization improved insulin sensitivity in obese animals with insulin resistance. In adipocyte cultures, resistin decreased glucose transport in response to insulin, while an anti-resistin antibody produced the opposite effect. What's more, expression of resistin decreases when given insulin, leptin or adiponectin and increase under an inflammatory situation.
Now, there exists strong argument of resource, effect and mechanism of resistin. First, it is once thought that white adipose tissue, especially that in organs, is the main source of resistin. However, some other studies found it is single nuclearcyte/M(?) makes most contribution to resistin excretion. There exist points that resistin is mainly secreted by preadipocytes other than mature adipocytes, meanwhile opposite points exist as well. A third point demanstrates that resistin expression first increase accompanied with preadipocytes differentiation, then begin to decrease after getting to a certain peak. Secondly, among hundreds of researches about relationship between resistin gene SNP and insulin resistance, all three possible results:positive relating, negative relating and no evident relationship exist at the same time. This is possiblely
due to different sample sizes and statical methods, however, all researches consider resistin as an important factor in insulin resistance and T2DM.
It is found that patients with obesity are accompanied by insulin resistance to some extend. Obesity is often considered as the situation with exccesive adipose tissue. Adipose-derived stem cell, as an essential part of adipose tissue, plays a vital role to adipose tissue through its poliferation and differentiation, and makes effect on the whole endocrine system.
So that, it is important to clarify the effect and mechanism of human resistin,which is one of keys to reveal effects put on adipocytes by resistin, and the relationship between resistin and insulin resistance. Those mechanisms are essential basement of new medical treatments.
In this study, we try to contribute a resistin-over expressing model to find out what effects would be put on adipose-derived stem cell within an environment of resistin overdose. Nowadays, most researches of resistin have a keen on directly putting recombined resistin in to cultured cells. It has been proved active resistin in vivo often exists as trimer or hexamer, however, recombined resistin through E.coli exists as dimer or single molecule. So that, the possibility that activity of recomined resistin is lower can not be neglected. Plus, most studies use 3T3-L1 cell line as the target cell, which is derived from mice, in which there exist many differences in resistin gene compared with human. What's more, ordinary transfection medium not only can be used in cells with strong proliferating ability but also brings injury to cells. To avoide such problems, we use lentivirus with resistin gene as a vector and use it to affect adipose-derived stem cell since lentivirus, as a vector, makes less injury to cells and can affect cells in stable stage with a persisting and stable expression.
Our study can be divided into 3 parts:1. Separation, culture, and identification of adult adipose-derived stem cells. (1) using immunochemistry to test cell surface marker CD90; (2) Using FCS to test cell surface marker CD 13 (3) transdifferenciating cells into adipocytes.2. Construction of expressing vector pWPXLd-hR. (1) use lipotom instantly transfecte 293F-T cell to make 3 lentivirus plasmids constructed into lentivirus vector (3) observe lentivirus affected cells under fluorescence microscope.3. Lentivirus with human resistin gene transfecting adipose-derived stem cell. (1) Using immunocytology to test resistin expression during different period of adipose-derived stem cells transfection into mature adipocytes. (2). Using immunocytology and oil red staining to test affections of resistin over-expression to shape, differentiation and proliferation ability of adipose-derived stem cells. Result:
1.In the adult adipose tissue there was a kind of fibroblast-shaped cells which could be stably cultured, passaged and differentiated into adipocytes in vitro. Compared with the blank control, both surface marker CD 13 and CD90 were positive in those cells.
2.Using genetic methods to re-construct lentivirus expressing vector with human resistin gene. After restriction enzame and DNA scequnce analysing, it is showed human resistin cDNA has been successfully transfered from PcDNA-hR into pWPXLd, with a correct orientation and sequence array, which implies lentivirus expressing vector with human resistin gene-pWPXLd-hR, the basement of coming experiments.
3.Construction of lentivirus with human resistin gene in vivo Successfully constructed 3 plasmids related with lentivirus:pWPXLd (10455bp) psPAX2 (10703bp) and pMD2.G (5824bp) into virus with inffection ability. After immunocytology test, it shows all host cells inffected do over-express resistin so that this virus is able to integrate target genes into host cells and make them expressed continuely.
4.During the differentiation process of adult adipose-derived stem cells into mature adipocytes, the expression of resistin decrease step by step but still slightly exists all the time.During the procedure that adipose-derived stem cells differentiating into mature adipocytes, the expression of resistin decreases and falls to a stable statement at last. Mature adipocytes still slightly express resistin.
After transfected with Lv-hR, adipose-derived stem cells lost their origenal shape and arrangement with granuls inside. The process of differentiating into adipocytes is obvious suppressed. However, number of the cells does not show any difference.
In all, our results indicated that three insulin target cells are all targets of human resistin and human resistin maybe induced periphery insulin resistance by different mechanisms. That hR maybe had no direct effect on insulin sensitivity of adipocytes, however, it suppressed adipogenesis so that the population of mature adipocytes were cut down, which led to impairment of insulin sensitivity. Our results revealed relationship between human resistin and insulin resistance, which demonstrated human resistin is close correlated to obesity and insulin resistance. Moreover, the results indicated effect of human resistin on insulin signal transduction pathway on cell level and molecular level. This establishs groundwork for further study medicine targeting on resistin curing T2DM and obesity.
Influnce of human resistin transfecting adult adipose-derived stem cell
Adipose tissue was once thought just as a resource of energy, however, it has been seen as an important endocrine tissue with more and more regards recently. It has been proved adipose tissue plays an essential role in regulating human metabolism through its secretion of kinds of protiens such as leptin, adiponectin, resistin, TNF-a ect. Resistin belongs to resistin-like molecules family (RELM), so that is also called FIZZ3. In human and rodents, resistin takes effect in tissues associated with glucose metabolism such as hepatic tissue, muscle and adipose tissue. In particular, administration of recombinant resistin to ob/ob mice, db/db mice and 3T3-L1 preadipocytes in vitro produced insulin resistance, whereas resistin immune neutralization improved insulin sensitivity in obese animals with insulin resistance. In adipocyte cultures, resistin decreased glucose transport in response to insulin, while an anti-resistin antibody produced the opposite effect. What's more, expression of resistin decreases when given insulin, leptin or adiponectin and increase under an inflammatory situation.
Now, there exists strong argument of resource, effect and mechanism of resistin. First, it is once thought that white adipose tissue, especially that in organs, is the main source of resistin. However, some other studies found it is single nuclearcyte/M(?) makes most contribution to resistin excretion. There exist points that resistin is mainly secreted by preadipocytes other than mature adipocytes, meanwhile opposite points exist as well. A third point demanstrates that resistin expression first increase accompanied with preadipocytes differentiation, then begin to decrease after getting to a certain peak. Secondly, among hundreds of researches about relationship between resistin gene SNP and insulin resistance, all three possible results:positive relating, negative relating and no evident relationship exist at the same time. This is possiblely
due to different sample sizes and statical methods, however, all researches consider resistin as an important factor in insulin resistance and T2DM.
It is found that patients with obesity are accompanied by insulin resistance to some extend. Obesity is often considered as the situation with exccesive adipose tissue. Adipose-derived stem cell, as an essential part of adipose tissue, plays a vital role to adipose tissue through its poliferation and differentiation, and makes effect on the whole endocrine system.
So that, it is important to clarify the effect and mechanism of human resistin,which is one of keys to reveal effects put on adipocytes by resistin, and the relationship between resistin and insulin resistance. Those mechanisms are essential basement of new medical treatments.
In this study, we try to contribute a resistin-over expressing model to find out what effects would be put on adipose-derived stem cell within an environment of resistin overdose. Nowadays, most researches of resistin have a keen on directly putting recombined resistin in to cultured cells. It has been proved active resistin in vivo often exists as trimer or hexamer, however, recombined resistin through E.coli exists as dimer or single molecule. So that, the possibility that activity of recomined resistin is lower can not be neglected. Plus, most studies use 3T3-L1 cell line as the target cell, which is derived from mice, in which there exist many differences in resistin gene compared with human. What's more, ordinary transfection medium not only can be used in cells with strong proliferating ability but also brings injury to cells. To avoide such problems, we use lentivirus with resistin gene as a vector and use it to affect adipose-derived stem cell since lentivirus, as a vector, makes less injury to cells and can affect cells in stable stage with a persisting and stable expression.
Our study can be divided into 3 parts:1. Separation, culture, and identification of adult adipose-derived stem cells. (1) using immunochemistry to test cell surface marker CD90; (2) Using FCS to test cell surface marker CD 13 (3) transdifferenciating cells into adipocytes.2. Construction of expressing vector pWPXLd-hR. (1) use lipotom instantly transfecte 293F-T cell to make 3 lentivirus plasmids constructed into lentivirus vector (3) observe lentivirus affected cells under fluorescence microscope.3. Lentivirus with human resistin gene transfecting adipose-derived stem cell. (1) Using immunocytology to test resistin expression during different period of adipose-derived stem cells transfection into mature adipocytes. (2). Using immunocytology and oil red staining to test affections of resistin over-expression to shape, differentiation and proliferation ability of adipose-derived stem cells. Result:
1.In the adult adipose tissue there was a kind of fibroblast-shaped cells which could be stably cultured, passaged and differentiated into adipocytes in vitro. Compared with the blank control, both surface marker CD 13 and CD90 were positive in those cells.
2.Using genetic methods to re-construct lentivirus expressing vector with human resistin gene. After restriction enzame and DNA scequnce analysing, it is showed human resistin cDNA has been successfully transfered from PcDNA-hR into pWPXLd, with a correct orientation and sequence array, which implies lentivirus expressing vector with human resistin gene-pWPXLd-hR, the basement of coming experiments.
3.Construction of lentivirus with human resistin gene in vivo Successfully constructed 3 plasmids related with lentivirus:pWPXLd (10455bp) psPAX2 (10703bp) and pMD2.G (5824bp) into virus with inffection ability. After immunocytology test, it shows all host cells inffected do over-express resistin so that this virus is able to integrate target genes into host cells and make them expressed continuely.
4.During the differentiation process of adult adipose-derived stem cells into mature adipocytes, the expression of resistin decrease step by step but still slightly exists all the time.During the procedure that adipose-derived stem cells differentiating into mature adipocytes, the expression of resistin decreases and falls to a stable statement at last. Mature adipocytes still slightly express resistin.
After transfected with Lv-hR, adipose-derived stem cells lost their origenal shape and arrangement with granuls inside. The process of differentiating into adipocytes is obvious suppressed. However, number of the cells does not show any difference.
In all, our results indicated that three insulin target cells are all targets of human resistin and human resistin maybe induced periphery insulin resistance by different mechanisms. That hR maybe had no direct effect on insulin sensitivity of adipocytes, however, it suppressed adipogenesis so that the population of mature adipocytes were cut down, which led to impairment of insulin sensitivity. Our results revealed relationship between human resistin and insulin resistance, which demonstrated human resistin is close correlated to obesity and insulin resistance. Moreover, the results indicated effect of human resistin on insulin signal transduction pathway on cell level and molecular level. This establishs groundwork for further study medicine targeting on resistin curing T2DM and obesity.
引文
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