The Research About The Relationship Between SCP2Gene And The Lithogenicity Of Bile In Mouse Model

1. Expression of mouse SCP2gene adenoviral vector carrying albumin promoter in Hepal-6cellsObjectives:We constructed the replication defective adenoviral vector of SCP2gene carrying murine albumin promoter, and studied its expression level in Hepal-6cell.Methods:The cDNA of SCP2gene was cloned by using RT-PCR technique. The albumin promoter was linked to SCP2gene’s upstream, and the GFP gene lied in its downstream. The plasmid PDC312-ALB-SCP2-IRES2-EGFP was constructed by the gene recombination technique, and the vector was identified with the ways of PCR, enzyme digestion and sequencing. The Admax Adenoviral Vector System was used to generate the replication defective adenoviral vectors, which were purified by CsC1method. The processes of TCID50were applied to detect the titers of the adenoviral vectors. The RNA and protein were respectively extracted from the infected Hepal-6cells by the adenoviral vector. The real-time quantitative PCR was employed to detect the mRNA expression levels, and the Western blot analysis was used to measure the SCP2protein levels.Result:(1) We constructed successfully the replication defective adenoviral vector of SCP2gene carrying albumin promoter.(2) In Hepal-6cells of mice, SCP2mRNA levels add about170times (t=96.91, p<0.05), and SCP2protein expression increase about9times (t=88.73,p<0.05) compared with controls; their differences are statistically significant.(3) When the mRNA levels of SCP2gene were overexpressed, CYP7al mRNA levels were down-regulated about2times (t=3.97, p<0.05); but the mRNA levels of HMGCR up-regulated2times (t=3.23, p<0.05).Conclusions:We constructed successfully the replication defective adenoviral vector of SCP2gene carrying albumin promoter. The SCP2gene overexpression may give rise to the variation of HMGCR and CYP7algenes in mRNA levels and affect cholesterol and bile acid metabolism, which could promote the formation of cholesterol calculus.2. Construction and identification of the replication defective adenoviral vectors carrying mouse SCP2-ShRNAObjectives:It was constructed that the replication defective adenoviral vectors carried the short hairpin sequences of mouse SCP2.And we will make a further study of mechanism between SCP2gene and cholesterol stone in gallbladder. Methods:The short hairpin sequences of mouse SCP2were cloned by two-step PCR, and connected together with the plasmid pDC312.The Admax Adenoviral Vector System was used to generate the replication defective adenoviral vectors, which were purified by CsCl method. The processes of TCID50were applied to detect the titers of the adenoviral vectors. Furthermore, Protein levels of SCP2were determined by Western blot analysis,and the levels of SCP2、CYP7al、HMGCR mRNA from the hepal-6cell of mouse were measured by the usage of RT-PCR.Results:(1) SCP2mRNA and SCP2protein were down-regulated by the replication defective adenoviral vectors carried the SCP2-shRNA.(2) Compared with the control group Adssh, the difference of both Adshl and Adsh2are statistically significant (p<0.05). Moreover, the inhibition rate of Adsh1and Adsh2to SCP2mRNA is respectively83.5%and71.6%. Both Adshl and Adsh2have silent effects, but the former inhibition effect is better.(3) When SCP2gene expression levels low, CYP7al mRNA expression increase about2times (t=10.10, p<0.05), and HMGCR mRNA expression drop more than twice times (t=10.68, p<0.05) compared with control group.Conclusions:The replication defective adenoviral vectors carried SCP2-shRNA were constructed successfully. The lower levels of SCP2could affect the activities of HMG-CoA reductase and CYP7al enzyme, which change cholesterol metabolism and could prevent the formation of cholesterol stone.3. SCP2gene involves the formation of the cholesterol stone by affecting cholesterol and bile metabolism.Objective:The mice were respectively injected normal saline, the empty virus, the replication defective adenoviral vectors carried SCP2gene and the short hairpin sequences of SCP2gene to make a further study of mechanism between SCP2gene and cholesterol stone in gallbladder.Methods:(1) The biliary drainage model of mouse was constructed. After the mouse narcotized, the abdomen was cut open to find duodenal papilla and bravery manager openings. PE10tube was retrograde inserted bravery manager by duodenal wall, then bile flow can be observed if intubation tube succeeded.(2) At8weeks of age, forty C57BL/6mice (male and female half) were divided into4groups at random, with each group containing10mice. The4groups mice were respectively injected normal saline, the empty virus, the SCP2gene overexpression and the SCP2shRNA replication defective adenoviral vectors.(3) Hepatic bile was collected for up to90 min by abovementioned model and calculate bile blow rate. Bile samples were stored at-80℃.(4) High performance liquid chromatograph (HPLC) was employed to measure cholesterol、phospholipid and cholate of bile and calculate the lithogenic index (LI).(5) Realtime quantitative PCR was used to detect mRNA expression levels of SCP2、HMGCR、ABCG5、ABCG8、ABCB11、CYP7al and PKC-b genes.Result:There are no differences between physiological saline group and empty virus group. Compared with empty virus group, cholesterol、phospholipid and cholate of bile and lithogenic index of the SCP2overexpression group are all increased, while bile blow rate is discreased. On the contrary, those of the SCP2shRNA group have an opposite tendency, the differences are statistical significance (p<0.05). To the SCP2overexpression group, SCP2mRNA levels raise about4times (p<0.05), HMGCR mRNA levels unregulated morn than2.5times (p<0.05), CYP7al mRNA levels down-regulate over2times (p<0.05), both of ABCG5and ABCG8gene’s expression levels enhance about1.5times, but the former is not statistical significance (p>0.05), the latter is statistical significance (p<0.05); ABCB11mRNA levels elevate about1.7times (p<0.05), compared with empty virus group. On the contrary, to the shRNA group, SCP2mRNA levels reduce about3times (p<0.05), HMGCR mRNA levels down-regulated about2times (p<0.05), CYP7al mRNA levels elevate less than2times (p<0.05), ABCG5mRNA levels lower about1time (p>0.05), ABCG8gene’s expression levels also decline about1time (p<0.05), ABCB11mRNA levels are more than half (p>0.05), compared with the control group.Conclusion:when SCP2gene is overexpressed,(1) cholesterol transit ability increases, and the cholesterol transported to liver also increases accordingly;(2) HMGCR enzyme activity enhanced, lead to the synthesis of cholesterol accelerated in the liver, so the cholesterol concentration of bile increase corresponding;(3) ABCG5and ABCG8gene expression levels are elevated, which cause the secretion of bile cholesterol increased;(4) The added ABCB11mRNA levels may make the secretion of phospholipids and cholesterol increase in bile, duing to some kind of particular feedback adjustment or compensation mechanism;(5) CYP7al enzyme activity is reduced, so bile acid synthesis abilities drop, and the discharge of cholesterol will diminish. The comprehensive function of these factors will easily lead to cholesterol supersaturation and crystallization in bile, and finally promote cholesterol gallstone formation. However, when SCP2gene is knocked out by RNA interference method, these factors play the opposite effects, which can prevent the formation of cholesterol calculus. In a word, SCP2gene involve the formation of the cholesterol stone as a result of it causing the cholesterol and bile metabolic changes.