Establishment Of CHO Cell Line Stable Expressing Human MCHR2 Gene And Construction Of Its ShRNA Eukaryotic Expressing Vector

Obesity is an ordinary trophopathy disease in the modern society. The incidence rate of it is rising year by year in both developed and developing countries, which becomes a global healthy problem. Obesity is not only an important factor that leads to cardia and brain vascular disorders, diabetes mellits, hypertension, hyperlipoidemia, but also brings us many social and psychological problems. After years' research, scientists discover that the occurrence of the obesity is closely related to the control of the neural endocrine of ingesting through the hypothalamus. Recently after the neuropeptide Y(NPY), agouti related protein(AgRP) and orexin(ORX) that can increase people's appetite, now another melanin-concentrating hormone (MCH)related to appetite enters people's sight. With the gradual deepening studying, the scientists discovered two MCHR in succession, namely, MCHR1(melanin-concentrating hormone receptor1, MCHR1) and MCHR2(melanin-concentrating hormone receptor 2, MCHR2). Many animal experiments prove that MCH is a kind of neuropeptide which can increase appetite and plays an important role in regulating the balance of the energy. By establishing a mouse with excessively expressing MCH, we find that it can ingest too much . At the same time, it has the characteristics of high lipoprotein blood atrophy, high blood glucose and resistance to insulin. On the contrary, a mouse without MCH gene may lead to thin syndrome, for example ,the mouse ingest less food at this time; its metabolism is increasing, moreover, the mouse become thinner. From the experiment we know that MCH plays a key role in regulating weight. We use the technology of gene knock-down to research the function of MCHR1, and find that the body is very thin with less fat. It is not easy to become fat by eating diet full of high fat. At present, the antagon of MCHR1 has been developed to cure obesity. There is no doubt that MCH and MCHR1 are related to the obesity and metabolism, but the function of MCHR2 is still not clear to us nowadays .What is more, there seems no clear report about it related to obesity whether at home or abroad.MCHR2 gene is located between chromosome 6q16.2 and 6q21 with the whole length of cDNA about 1023 bp, and codes the protein with 340 amino aids. By the technology of quantitive RT-PCR, Northern hybridization and in situ hybridization ,it is proved that MCHR2 mainly lies in cerebrum. However, the regulating main centre of ingesting lies in hypothalamus, which indicates that MCHR2 may control and regulate the ingestion. In addition, it is reported that the cell genetics of the obese patient has changed in chromosome 6q, and MCHR2 is just located in chromosome 6q. All above shows that MCHR2 is likely to be related to the occurrence of obesity. Therefore , it is necessary to make a further research about the function of MCHR2 gene so as to make sure of the relationship between it and obesity. Thus we can provide new targets for curing obesity.RNA interference ( RNAi ) is a process of gene silence conducted by double strand RNA. As an emerging gene blocking technology, it gradually becomes the new method to explore the function of gene .This experiment uses RNAi , targets human MCHR2 gene ,constructs shRNA eukaryotic expressing vector, and researches the effect that it has on the MCHR2 expression in cells and the characters of its biological activity .Meanwhile, it lays a good experiment foundation for using RNA interference technology to go on a research of MCHR2 gene function based on the average level of animals . Our research is mainly divided into 3 parts as follows :1: The establishment of CHO cell line stable expressing human MCHR2. Methods: we use the human fetal brain cDNA library as template to amplify the whole length of cDNA fragment of MCHR2 gene by PCR. Then we insert it into eukaryotic expression vector pcDNA3.1(+), after the identification of restriction endonuclease digestion and sequencing ,we transfect it into CHO cell by lipofectamineTM2000.At last, through the selection of G418 ,we can build a stable transfected CHO cells line. The MCHR2 expression can be tested by using RT-PCR,western blotting and immunofluorescence. We also use radioligand binding assay(RBA) , calcium influx assay and cyclic adenosine monophosphate(cAMP) to make a research of the molecular characteristics. Results: we construct the pcDNA3.1(+)/MCHR2 eukaryotic expressing vector successfully and establish the stable tranfected CHO cells line. Moreover, we successfully express the goal gene. The Bmax of it is 309.97±1.14 fmolL-1·mg-1protein and the dissociation constant is 0.170±0.0006 nmol/L. MCH can stimulate Ca2+ release, EC50 is 2.32±0.01 nmol/L. Conclusion: the construction of the stable transfected CHO cell line and the research of MCHR2 molecular characteristics have laid a good experiment foundation for further research about the function of MCHR2.2: The design of human MCHR2 shRNA and the establishment and identification of its eukaryotic expression vectors. Methods: according to MCHR2 gene sequence, we design and synthesize some siRNA, then we clone it to the eukaryotic expression vector pGenesil-1 which has kalamycin resistance and EGFP, then we have a restriction endonuclease digestion analysis and DNA sequencing on the reconstructed plasmid. Results: we successfully build 4 eukaryotic expression vectors expressing shRNA and its negative control plasmid. Conlusion:the establishment of human MCHR2 shRNA eukaryotic expression vectors has laid a foundation for further research of MCHR2 functions by RNAi.3: The effects of shRNA eukaryotic expression vectors on human MCHR2 gene expression and its characters. Methods: we transfect shRNA eukaryotic expression vectors to CHO cells which stably express human MCHR2 gene, examine the expression of mRNA for MCHR2 gene by RT-PCR, measure the changes of MCHR2 protein by western blotting, examine the changes of Bmax and Kd by RBA , we also use Calcium influx assay experiment to observe the release of Ca2+ in single cell and the EC50 changes. Results: shRNA eukaryotic expression vector can suppress MCHR2 gene expression in CHO cells effectively, compared with the pGenesil-1 empty vector, MCHR2 mRNA expression has reduced 45.8%～66.4%. Protein expression has reduced 44.2%～81.0%. In RBA, Bmax has reduced 39.4%～78.7%, while Kd increased 40.9%～81.9%. After the transfection of shRNA eukaryotic expression vector, we stimulate single cells with MCH, Ratio A has reduced, while EC50 goes up. Compared with the pGenesil-1 empty vector, EC50 increased 114.8%～822.4%. We conclude that shRNA eukaryotic expression vector can suppress MCHR2 gene expression in CHO cells effectively, at the same time, reduce Bmax and increase Kd and EC50.