The Research On Cloning Of LPL And The Extraction And Culture Of Adipocytes In Grass Carp

Body fat deposition of animals will cut down the percentage of lean meat in naked body and result in low-grade meat of animals. Our country′s output of aquatic products were the maximum all over the world. One of the main problems about breeding vocation is excessive accumulation of adipose tissue in fish. It is confirmed that lipoprotein lipase (LPL) that is critical lipase of lipolysis. It is one of the symbol genes during early period of lipolysis.The study discovered that LPL is an important functional protein that adjust the lipid metabolism of body by obesity genes of mammalians. LPL affects the state of lipid accumulation of body fat deposition determination.The research of adipocyte proliferation and differentiation and factors with regards to fat deposition is a project that has practical importances and economical senses. The essential method about reseach of the rule of animal fat deposition was adipocytes culturing in vitro and monitoring the process of adipocyte proliferation with reseaching about development regulation of adipose tissue.The biology of adipose tissue has identified the adipocyte as an important mediator in many physiologic and pathologic processes regarding energy metabolism. Excess of fat deposition leads to human obesity and related metabolism disease, also causes the decrease in quality of domestic productand the fishes. The proliferation and differentiation of adipocytes is a dynamic and plastic process under the influence of various hormones, genes and signaling pathway. Lipoprotein lipase(short for LPL)was the classical symbol gene of the early differentiation. Thus, grass carp, which was widely breeding in China, was used as the experimental animal. LPL cDNA sequence of grass carp was cloned and the pattern of tissue expression was investigated. Two types of LPL gene were found in grass carp:LPL1 and LPL2. Established the extraction ang culture system of grass carp SVF cells and adipocytes. Then, the research of SEM and TEM showed us the stereo-structure and intracellular micro-stucture of grass cqrp SVF cells and adipocytes were done.The main results were as follows:1. The length of LPL1 cDNA sequence cloned by PCR technology was 453 bp and the length of LPL2 cDNA sequence was 360 bp. Homologous alignment analysis using Clustal W software showed that grass carp LPL1 gene had 66%,62%,65%,68%and 67% homology to that of zebrafish, red sea bream ,pig, house mouse and Norway rat respectively, and the homology of LPL2 gene were 89%,73%,69%,70%and 70%. This identified that LPLwas evolutionarily conserved in history.2. The expression of LPL1 and LPL2 mRNA in various grass carp tissues from different developmental stages was investigated by Semi-quatitive RT-PCR. The results showed that LPL1 and LPL2 gene were widely expressed in heart, liver, spleen, gonad, kidney, muscle and visceral adipose from 500g yo 800g weight grass carps.Furthermore, the expression of LPL1 mRNA in heart tissue was significantly higher than other tissues, the expression of LPL2 mRNA in liver tissue was significantly higher than other tissues. Additionally, LPL1 mRNA and LPL2 mRNA in grass carp adipocytes was significantly higher than grass carp SVF cells(p<0.05).3. Established the extraction and culture system of grass carp SVF cells and adipocytes. The key factors were as follows: extraction conditions,2% collengnase ,digestion time as 30min to 45 min, the size of steel sieve was 100 eyes and 200 eyes and the centrifuge speed is 1500-2000rpm, centrifuge time is 10mins. The culture conditions were as follows: 25℃-28℃,3%CO2, and the DMEM/F12 (contained 10% FBS) were used as culture medium.4. The research of SEM and TEM showed us the stereo-structure and intracellular micro-stucture of grass carp SVF cells and adipocytes. This is the first time to discribe these cells'structure directly.