摘要
现有大量临床研究资料显示,高甘油三酯(TG)血症是冠心病(CHD)的独立危险因素,并有促动脉粥样硬化斑块破裂的危险。寻找高脂血症的发病原因,探讨高脂血症的预防和治疗已成为目前国内外学者研究的热点和难点,而选择理想的高脂血症动物模型则是研究脂质代谢紊乱的关键。
多年来,高TG血症动物模型多是通过给大鼠或小鼠喂饲高脂高糖饮食而造成。由于大鼠和小鼠对此类饮食敏感性差,且血浆脂蛋白的合成及清除率与人差异较大,所以很难作为理想的高脂血症动物模型来进行深入研究。兔也有广泛应用,但猪模型的优点在于解剖学和生理学与人类极为相似。
载脂蛋白CⅢ(ApoCⅢ)参与富含TG脂蛋白的生成。研究发现ApoCⅢ能抑制脂蛋白脂酶(LPL),而后者是清除血浆脂蛋白中甘油三酯的限速酶。血浆ApoCⅢ浓度与甘油三酯水平呈正相关。在转基因鼠研究中,大量表达人ApoCⅢ基因可以造成高甘油三酯血症。如果小鼠中ApoCⅢ基因被敲除则血浆TG水平只有正常小鼠的70%。
本研究采用DNA重组技术,构建重组ApoCⅢ真核表达载体,转染猪胎儿成纤维细胞,以G418筛选出阳性克隆细胞,经核移植、胚胎移植,最终有3只代孕母猪妊娠期满,顺利产下14只仔猪,成功获得10只ApoCⅢ转基因克隆猪,获得5只高脂血症模型。此研究对探讨相关疾病病因、发病机理和基因治疗方法,研究基因表达调控以及进行药物干预和新药的开发试验等,都将具有极为重要的意义。
初步表型分析结果表明,本研究建立的ApoCⅢ转基因克隆猪F0代,血浆TG水平是正常对照组的2-3倍,LPL活性下降2.4倍。
Apolipoprotein CⅢ(ApoCⅢ) plays an important role in plasma triglyceride rich lipoproteins and their remnant metabolism. ApoCⅢis generally considered to inhibit lipoprotein lipase (LPL) activity and receptor-mediated endocytosis of lipoprotein particles competitively by interfering with endothelial proteoglycans and specific lipoprotein receptor binding.
Mature ApoCⅢis a 79-amino acid glycoprotein that exists in chylomicrons (CM), very low density lipoproteins (VLDL) and high density lipoproteins (HDL). The ApoCⅢenhancer is involved in maintaining an active chromatin subdomain in the ApoAI/CⅢ/AⅣregion and regulates the liver-and intestine-specific expression of the three genes of the cluster, but not of ApoAV at the chromatin level.
Large-scale clinical trials have indicated that hypertriglyceridemia is an independent risk factor for coronary artery disease (CAD), and high levels of ApoCⅢare often correlated with hypertriglyceridemia. However, people with hereditary deficiencies in ApoCⅢhave lower plasma triglyceride levels, and carriers of a null mutation in ApoCⅢhave cardioprospective plasma lipid profiles with lower LDL-C and higher HDL-C. Overexpression of human ApoCⅢin transgenic mice leads to hypertriglyceridemia, and ApoCⅢknockout mice have reduced levels of plasma triglyceride-containing lipoproteins.
However, the lipoprotein profile of mice is different from that of humans, so that the mouse model is not suitable for evaluating the role of ApoCⅢin atherosclerosis or other cardiovascular diseases. To develop a model overexpressing human ApoCⅢand to investigate the role of ApoCⅢin lipid metabolism in a model organism more similar to humans, we generated a human-ApoCⅢtransgenic minipig model through nuclear transfer. The porcine model is appropriate for studying lipoprotein metabolism associated with hyperlipidemia because pigs have both LDL and HDL particles in plasma and a humanlike cardiovascular system. Experimental CAD induced in the porcine model is similar to its natural progression in human patients.
A 10-kb human genomic DNA fragment spanning the ApoCIII gene was successfully used to generate human-ApoCⅢtransgenic minipigs. Ten transgenic pigs were born and five founders survived. In this report, the five founders were used to analyze the phenotype of human ApoCⅢoverexpression in pigs. These pigs will be bred to yield a line of transgenic minipigs for further study.
In this study, we successfully produced transgenic miniature swine expressing human ApoCⅢ. A total of 1600 reconstructed embryos were transferred into 8 recipients following natural estrus. Six of them showed early pregnancies and three of them farrowed down. Fourteen male pups were born through natural delivery on days 113-115, including two dead fetuses. Five of them died 1-10 days after birth for unknown reasons, and the remaining swine (five transgenic and two nontransgenic) survived and grew healthily.
Transgenic swine showed a significant increase in plasma triglyceride (TG) levels, with 2.5-fold (94.9392±10.3838 vs.38.3721±3.4748 mg/dl) and 2.3-fold (40.6573±14.2967 vs. 17.5017±1.59 mg/dl) increases of TG in the postprandial and fasting states compared with control swine. Total cholesterol and HDL-C levels were slightly elevated in transgenic swine.
The TG levels in transgenic swine took longer to reach peak level in an oral fat-load test (2.5 hours vs.2 hours), and the peak level was 2.3 times greater than in control swine (88 mg/L vs.39 mg/L). This result suggests that transgenic swine have delayed triglyceride absorbance and clearance.
Overexpression of human ApoCⅢin swine resulted in a 2.4-fold decrease in LPL activity in post-heparin plasma (103.7±31.0 vs.249.7±69.2 mU/ml), compared with wild type.
Clinically, plasma ApoCⅢlevels were associated with increased plasma triglycerides, and the human ApoCⅢnull mutation led to low plasma triglycerides, so that human ApoCⅢtransgenic and knockout animal models will be the preferred tools for studying the mechanism of hypertriglyceridemia-associated diseases and drug development. In the present study, we generated human-ApoCⅢtransgenic swine and analyzed the phenotype of this model with respect to lipid metabolism. Although the genetic modification of somatic cells followed by nuclear transfer is inefficient, no other techniques can be utilized in swine or other livestock for generating knockout and transgenic animals. We followed this strategy for human-ApoCⅢminiature swine and proved that the human ApoCⅢgene had been integrated into the swine genome and expressed in the liver and intestine. We have also generated the same gene transgenic model by zygote microinjection in mouse and rabbit germ lines. The overexpression of human ApoCⅢin all of these transgenic animals resulted in hypertriglyceridemia of varying degrees.
Generally, swine are the preferred animal model for cardiovascular disease because they have a humanlike cardiovascular system. In this study, the plasma triglyceride levels of human-ApoCⅢtransgenic miniature swine reached 110-130 mg/dl on a chow diet; however, the levels were relatively low (about 80 mg/dl). This feature is the same as in the transgenic rabbit model (about 190 mg/dl), but mice differ markedly from swine and rabbits in this respect (300 mg/dl～1000 mg/dl). Hence, the swine and rabbit models more closely mimic human plasma lipids, because clinical hypertriglyceridemia commonly displays a light or medium increase in triglyceride levels.
It is possible that differences between species and integration sites explain some of the discrepancies between plasma TG levels in transgenic minipigs and transgenic mice. The markedly delayed clearance of olive oil and reduced plasma LPL activity in transgenic miniature swine suggest that the elevation of triglycerides may be due to the impairment of triglyceride hydrolysis by LPL. This finding is consistent with the traditional view of ApoCⅢas a noncompetitive inhibitor of LPL
The swine model shows similar lipoprotein factions in plasma as human-carrying cholesterol, mainly in LDL and HDL lipoprotein particles. Our data indicate that hypertriglyceridemia of transgenic swine was due to an increase in circulating VLDL and chylomicron-sized particles, which contained more triglycerides. In light of the similar susceptibilities of humans and swine to atherosclerosis, further studies using the swine model may enable a better understanding of the role of triglycerides in atherosclerosis as well as the physiological effects of ApoCⅢon atherosclerosis.
In conclusion, we successfully generated transgenic miniature swine expressing human ApoCⅢ. Our study should provide a powerful model to gain further insight into the in vivo functional roles of human ApoCⅢon lipid metabolism and of triglycerides on atherosclerosis or CAD. Human-ApoCⅢtransgenic miniature swine are a better model than other germlines for evaluating the efficacy and pharmacology of new drugs for hypertriglyceridemia.
引文
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