基因重组融合蛋白hNPY对小鼠前脂肪细胞增殖和分化影响的实验研究

目的：前脂肪细胞是一类具有增殖和向脂肪细胞分化潜力的特异化的前体细胞，如能找到可促进前脂肪细胞增殖和分化的因子或药物，并在脂肪组织移植的同时使用，则有希望提高脂肪组织的移植效果，本实验探索一种新的人体肥胖关联基因和外周脂肪细胞激动剂一基因重组融合蛋白hNPY对小鼠3T3-L1前脂肪细胞增殖和分化的影响及其分子调控机制。方法：采用基因工程技术设计hNPY基因上下游序列，经过PCR反应合成hNPY的cDNA后与pET28a＋载体重组，再将已构建好、并经测序确认无误的重组质粒pET28a—NPY转导至大肠杆菌BL21（DE3），再由IPTG诱导表达hNPY融合蛋白、并进行纯化；然后，将体外培养的小鼠3T3-L1前脂肪细胞经由3-异丁基-1-甲基黄嘌呤、胰岛素和地塞米松进行联合诱导；诱导2d后，再将此细胞分为三组，即空白对照组（未加任何诱导剂组）、经典诱导组（胰岛素诱导）和实验干预组（hNPY融合蛋白干预），其中，实验干预组再按照hNPY融合蛋白浓度不同又分为高、中、低三个浓度组（10^-8mol／L、10^-19mol／L和10^-9 mol／L）。分别于细胞培养的第7d和第12d用相差显微镜观察各组细胞的形态学变化，再于细胞培养的第12d用油红O染色观察脂肪细胞的分化程度；同时，采用MTT法检测该细胞的增殖状况；采用Westernblot法检测脂肪细胞分化相关基因过氧化物体增殖剂活化受体-γ（PPAR-γ）、CAAT／增强子结合蛋白-a（C／EBP-a）蛋白的表达水平。结果：低浓度（10^-10 mol／L）的hNPY融合蛋白，无明显促进小鼠3T3-L1前脂肪细胞增殖和分化的作用效果；中浓度（10^-9 mol／L）的hNPY融合蛋白，可有效促进小鼠3T3-L1前脂肪细胞增殖及细胞数量增多；而高浓度（10^-8 mol／L）的hNPY融合蛋白，不仅能明显促进小鼠3T3-L1前脂肪细胞的细胞增殖和分化，且能显著提高该细胞C／EBPa和PPARγ的表达水平、而明显降低INSIG-2的表达。结论：高浓度（10^-8mol／L）的基因重组融合蛋白hNPY能够明显促进小鼠3T3-L1前脂肪细胞的增殖和分化，其促进3T3-L1细胞增殖和分化的分子调控机制很可能与上调PPARγ、C／EBPa表达和降低INSIG-2表达水平有关，这提示：hNPY作为脂肪细胞膜上受体NPYR的有效促进剂或激动剂，未来很可能成为人体外周脂肪细胞一个新的作用靶点。

Study on the Influence of Recombinant Fusion Protein hNPY on Proliferation and Differentiation of Mouse Preadipocytes

OBJECTIVE： To explore the influence and molecular regulation mechanism of recombinant fusion protein hNPY （a new human obesity gene and agonist of peripheral fat cells ） on proliferation and differentiation of mouse 3T3 - L1 preadipocytes. METHODS ： The upstream and downstream sequence of hNPY gene were designed by genetic engineering technology, cDNA of hNPY and pET28a ＋ vector after PCR reaction of synthesize cDNA were recombinanted, the recombinant plasmid pET28a - NPY which had been well constructed and sequentially confirmed was transplanted into E. coil BL21 （DE3） and induced by IPTG to express fusion proteins, then the expressed product was purified; In vitro 3T3 - L1 preadipocytes were induced to differentiation by the cocktailmedium containing 3 -isobutyl - 1 -methylxantine （IBMX）, dexa - methasone and insulin; After 2 days, the cells were divided into 3 groups： the control group （without inducer）, the routine group （insulin used as inducer） and the experiment group （fusion protein hNPY used as inducer）. Among the groups, the experiment group according to fusion protein hNPY was divided into high, moderate and low concentrations groups （10-8mol/L, 10-9mol/L and 10^-10 mol/L）. At day 7 and day 12 the morphological changes of cells were observed by phasecontrast microscope. At day 12 oil red 0 staining was performed to observe adipocyte differentiation; Proliferation capacities of 3T3 - L1 cells were assessed by MTT; Western blot was used to know the proteins expression level of peroxisome proliferator - activated receptor gamma （ PPAR - γ） and the CAAT/enhancer binding protein - a （ C/EBP - a）. RESULTS ： Low concentration （10^-10 mol/L） of fusion protein hNPY had no significant promotion effect on proliferation and differentiation of mouse 3T3 -L1 preadipocyte; Moderate concentration （10-9 mol/L） of fusion protein hNPY could effectively promote the mouse 3T3 -L1 preadipocytes proliferation and increase in the number; High concentration （10-s mol/L） of fusion protein hNPY not only significantly promoted cell proliferation and differentiation of mouse 3T3 - L1 preadipocytes but also could significantly improve the expression levels of C/EBP - a and PPARγ, but it could significantly reduce the expression levels of INSIG - 2 in 3T3 - L1 preadipocytes. CONCLUSION： High concentration （10^-8 mol/L） of fusion protein hNPY can significantly promote cell proliferation and differentiation of mouse 3T3 - LI preadipocytes. The molecular regulation mechanism of it on proliferation and differentiation of mouse 33T - L1 preadipocytes may he related to increased expression levels of PPARγ and C/EBPc（ and reduced expression levels of INSIG - 2, which suggests that hNPY is likely to become a new target of human peripheral adipocytes and an effective accelerator or agonist of adipocytes membrane receptors NPYR.