【作者】 王鹏；

【导师】 方定志；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 为研究代谢综合征(metabolic syndrome,MS)与动脉粥样硬化等疾病之间的关系，本研究室曾通过高糖膳食构建了高甘油三酯血症大鼠模型，其具有代谢综合征的基本特征。用该大鼠模型肝脏基因的差异表达序列片段筛选人肝脏cDNA文库后发现一条新的基因，国内外尚未有关于该基因的报道。该新基因被暂时命名为代谢综合征相关新基因(metabolism syndrome-related novel gene,MSRG)。为进一步了解MSRG与MS的关系，研究MSRG的功能，我室正在对影响MSRG转录因素进行系列研究。本文以HepG2细胞为研究对象，通过设计MSRG、β-actin引物及相应的Taqman探针，建立了RealtimePCR定量检测基因转录水平的方法。对HepG2细胞内MSRG转录水平进行检测后，发现MSRG相对于β-actin为低转录基因。我们用天然高密度脂蛋白(native high density lipoprotein,N-HDL)及氧化修饰高密度脂蛋白(oxidized high density lipoprotein,OX-HDL)孵育HepG2细胞，观察对细胞MSRG转录的影响。结果发现：①在N-HDL 0～100mg/L组及OX-HDL 0～50mg/L组，MSRG转录随N-HDL及OX-HDL浓度的增加有下降趋势。与0mg/L组相比，MSRG在N-HDL 50mg/L、100mg/L组和OX-HDL 50mg/L组转录水平差异具有统计学意义(p＜0．05)。当浓度高于此范围，MSRG转录较最低水平有所升高。与N-HDL 100mg/L组相比，N-HDL 200mg/L组基因转录有所回升，但无统计学差异(p＞0．05)；与OX-HDL 50mg/L组相比，OX-HDL 100mg/L、200mg/L组MSRG mRNA含量有增高趋势，且200mg/L组与50mg/L组间基因转录水平差异具统计学意义(p＜0．05)。②在N-HDL 0～24h组及OX-HDL 0～12h组，MSRG转录随时间延长有下降趋势。与0h相比，N-HDL 12h、24h、36h组基因转录降低(p＜0．05)，24h组MSRG mRNA含量最低；OX-HDL 6h、12h组基因转录较其0h组降低(p＜0．05)，12h组MSRG转录水平最低。当时间超过相应范围后，MSRG转录有回升倾向。与N-HDL 24h组相比，MSRG在N-HDL36h组转录有所回升，但无统计学差异(p＞0．05)；OX-HDL 24h、36h组MSRG转录较其12h组有所回升(p＜0．05)。③分别用100mg/L N-HDL或OX-HDL孵育HepG2细胞12h后，MSRG转录水平均较对照组显著下降(p＜0．05)，但二者间无显著差异。N-HDL、OX-HDL组培养基总胆固醇浓度较孵育前下降，甘油三酯浓度较孵育前上升，但均无统计学差异(p＜0．05)。细胞内糖原、总胆固醇及甘油三酯浓度在三组间均无显著性差异。我们发现：在低浓度及短时间内，N-HDL及OX-HDL均可降低HepG2细胞MSRG的转录水平。N-HDL较OX-HDL作用更为明显，抑制转录作用的浓度及时间范围均宽于OX-HDL。培养基由于加入了高密度脂蛋白，MSRG转录下降的同时伴有培养基胆固醇、甘油三酯浓度的改变。细胞内糖原、总胆固醇和甘油三酯浓度未发生显著变化。N-HDL及OX-HDL影响HepG2细胞MSRG转录的机制尚需进一步研究。更多还原

【Abstract】 A novel gene was cloned and temporarily named as metabolism syndrome-related gene (MSRG). To understand the relationship between MSRG and MS and the function of MSRG, our lab has been doing series of studies on the factors which can affect the transcription of MSRG. In the present study, we firstly established the method for quantification of the mRNA level of MSRG in HepG2 cells and found that MSRG, compared with the mRNA level ofβ-actin, was a lowly transcribed gene. Then, we investigated the effects of native high density lipoprotein (N-HDL) and oxidized high density lipoprotein (OX-HDL) on the transcription of MSRG in HepG2 cells. When HepG2 cells were incubated with 0mg/L, 25mg/L, 50mg/L, and 100mg/L of N-HDL or 0mg/L, 25mg/L, and 50mg/L of OX-HDL, the transcription of MSRG in the cells had a tendency to decrease with the increase of the lipoproteins. Compared with that in the cells incubated with 0mg/L of N-HDL, the transcription level was significantly decreased in the cells that were incubated with 50mg/L and 100 mg/L of N-HDL (p＜0.05). There was also a significant decrease of the transcription of MSRG in HepG2 cells, which were incubated with 25mg/L and 50mg/L OX-HDL, compared with that in cells incubated with 0mg/L of OX-HDL. Although not significantly, when compared with that in cells incubated with 100mg/L N-HDL, the transcription level was increased in the cells incubated by 200mg/L of N-HDL. The transcription in the cells incubated with 100mg/L or 200mg/L of OX-HDL was higher than that in cells incubated with 50mg/L of OX-HDL(p＜0.05). Incubated with 100mg/L N-HDL for Oh, 6h, 12h, and 24h or with 100mg/L OX-HDL for Oh, 6h, and 12h, the cells had a decreasing tendency to transcribe MSRG. The transcription in the cells, cultured by N-HDL for 6h, 12h, 24h, and 36h, was lower than that in cells incubated by N-HDL for 0h(p＜0.05). Compared with that in the cells cultured by OX-HDL for Oh, the transcription was decreased in the cells incubated with OX-HDL for 6h and 12h(p＜0.05). Between the cells incubated with N-HDL for 36h and exposed to N-HDL for 24h, the difference of MSRG transcription was not significant(p＞0.05). The transcription was significantly increased in the cells incubated with OX-HDL for 24h and 36h, compared with that in cells cultured by OX-HDL for 12h(p＜0.05). After incubation with 100mg/L N-HDL or OX-HDL for 12h separately, the levels of MSRG in the cells incubated with N-HDL or OX-HDL were decreased significantly compared with control (p＜0.05). The transcription of MSRG in the cells between the N-HDL and OX-HDL groups were not significant. In the medium, the levels of total cholesterol and triglyceride in N-HDL group and the level of total cholesterol in OX-HDL group were increased compared with control (p＜0.05). The levels of glycogen, total cholesterol, and triglyceride in cells were not significantly changed among control, N-HDL, and OX-HDL groups.The results suggest that both N-HDL and OX-HDL can influence the transcription of MSRG in HepG2 cells. When incubated with N-HDL or OX-HDL, the transcription of MSRG had a tendency to decrease with the increasing of lipoprotein concentration and length of lipoprotein incubation time. After reaching the lowest level, the transcription of MSRG had a tendency to increase with the continuous rising of lipoprotein concentration and incubation time. The ability of N-HDL to inhibit the transcription of MSRG was stronger than OX-HDL.更多还原