| BackgroundIt’s well known that the rupture of plaque surface in atherosclerotic (AS) is the major major pathological basis and pathogenesis of acute coronary syndromes (ACS), and the process of AS plaque from stable to vulnerable is related to inflammation, immunity, metabolism, coagulation and some other links. Thickness, strength and the content of collagen of lipid fibrous cap in AS plaque are crucial factors that determine the stability of AS plaque. Major component of the fibrous cap is extracellular matrix, which is made up by fibrous structural proteins, polysaccharides and proteoglycans. More thicker, more collagen fiber content in the fibrous cap, then the plaque less likely to rupture. Therefore, the rupture of fibrous cap is closely related to the reduction of ECM in fibrous cap. The matrix metalloproteinase (MMPs) is the most important enzymes to degrade extracellular matrix (ECM), with the degradation of extracellular matrix can make the AS plaque changes from stable to unstable, which can lead to plaque rupture and cause acute coronary syndrome.In human body, the CD147was named extracellular matrix metalloproteinase inducible factor (EMMPRIN), or known as basigin, M6, Neurothelin.which was early separated from lung cancer LX-1cells. It is a high glycosylation and widely expressed in hematopoietic and non-hematopoietic cell lines the transmembrane glycoprotein, belonging to a member of the immunoglobulin superfamily.Study show that CD147can induce the produce of the matrix metalloproteinase in smooth muscle cells, endothelial cells, monocytes and other cells, including MMP-1, MMP-2and MMP-3, MMP-9, and type1transmembrane matrix metalloproteinase protein (MT1-MMP). As may be gathered from this, CD147may play an important role in plaque’s stability by affecting the expression of MMPs.RNA interference means transmitting exogenous or endogenous dsRNA (double stranded RNA, dsRNA) into cells, causing the specific degradation of its cognate mRNA, leading to the unexpression of gene. Since the dsRNA can inhibit the expression of different targeted gene, RNAi technology has been aptly called gene knock out or gene silencing. As RNAi technology is the most simple and efficient way to block the expression of specific gene in mammalian cells, it’s also known as post-transcriptional gene silencing (PTGS), for it’s occurred posttranscriptional (PTGS). As the study deepening, it was found that the role of RNAi was not only in the post-transcriptional level, but also can be achieved in different levels of transcription and translation. The process of RNA interference caused by dsRNA in the human body can be divided into the two stages:Long-chain dsRNA can be identified and processed into21-23nucleotide small interfering RNA (siRNA) by Dicer(a nuclease activity of human1Rnase Ⅲ), Subsequently, these double-stranded siRNA was binding to the protein formation complex of RNA-induced silencing complex (RISC); Helicase in RISC unlock the double-stranded siRNA,and the latter bind to the highly complementary sequences mRNA through the unlocked antisense strand, guide siRNA and mRNA together. Finally, the siRNA and mRNA transposed in the complex, nuclease Dicer cut the mRNA into21-23nucleotide fragments, which can specifically inhibit the expression of target genes. The new double-stranded RNA fragments can be reformed of the RISC complex, continuing the degradation of mRNA and resulting the cascade effect. Therefore, each cell only needs several siRNA molecules but can cause a strong RNAi effect. Although some measures were successfully taken to prevent or reduce the occurrence and development of coronary heart disease, the existing medical and surgical methods is so limited to prevent and cure vulnerable or ruptured plaque. Various of currently used treatment methods, such as thrombolysis, lipid-lowering have some efficacy to ACS, but for the vulnerable or ruptured plaque repair is so limited. How to stabilize the vulnerable plaque are still the bottlenecks and difficulties in the prevention and treatment of ACS. In recent years, gene silencing techniques in therapy of cardiovascular disease has demonstrated the incomparable superiority compared with the traditional treatment method. It is realized if we can use RNAi technology to stabilize atherosclerotic plaque, which open up a new way for the treatment of cardiovascular disease. Along with advanced study on the mechanism of unstable plaques in atherosclerotic, matrix metalloproteinase and CD147molecules play an important role in unstable plaques. Inhibiting the expression and activity levels of MMPs and CD147makes significance on stabilize the plaque and prevent coronary events. The use of RNA interference technology with silence genes has broad application prospects.PART1The silencing effect of CD147lentiviral vector on rabbit peripheral blood monocytes-macrophage cellObjectiveCD147was named extracellular matrix metalloproteinase protease inducible factor in human body. It may play an important role on plaque stability by affecting the expression of MMP in smooth muscle cells, endothelial cells, monocytes/macrophages and other cells. To design and identify lentiviral vector RNA that specifically suppresses extracellular matrix metalloproteinase inducer (CD147) expression, and to screen those which can effectively suppress CD147expression in rabbit peripheral blood monocytes-macrophage cell.Methods1. Rabbit peripheral blood mononuclear cells were isolated and cultured Collect rabbit arterial blood and plus with heparin, diluted with equal amount of PBS with1:1ratio mix. Adding6ml monocytes separation medium into the bottom of15ml centrifuge tube, heparinized blood and equivalent PBS was superimposed on the stratified liquid, to keep the clear interface, horizontal centrifugate. Mononuclear cells were slowly separated from the second layer interface of the cloud layer with a sharp pipette. Add more than five times volume of preheated PBS solution, centrifugating and washing. After last centrifugation, the supernatant was removed and inoculated in50ml culture bottle, with37℃,5%CO2incubator for2h, and then aspirate the medium, wash three times with warm-up RPMI1640, non-adherent cells were eluted, and the remaining adherent cells are monocytes.2. Induced peripheral blood monocytes to macrophages. After these cells still for12h, to buy in complete medium RPMI-1640containing phorbol ester, stimulating for48h into macrophages.3. There were six groups:①control group:only add Polybrene2μl, with no treatment;②NC control group:add with NC lentiviral vector0.1ml and Polybrene2μl;③The CD147lentiviral interference groups (A、B、C、D):add with BSG-836, BSG-311, BSG-191, BSG-859lentiviral vector0.1ml and Polybrene2μl respectively, each group was setted with three replicates.4.72hours after monocytes and macrophagocytes were transfected into shRNA, the transfection results were observed by fluorescence microscopy. The expression of CD147mRNA and protein was assayed by fluorescence quantitative RT-PCR and ELISA, respectively. Observed expressions of CD147mRNA and protein by RNAi.5. Statistical analysis:Using SPSS13.0software for the statistical description and analysis of the data, the measurement data was shown with (x±s), If the overall variance is homogeneous, multiple sets of samples were compared with One-way ANOVA, Multiple compared using the LSD method test. If the overall variance is heterogeneous, using multiple independent samples non-parametric to analysis the Kruskal-Wallis H test. Comparing between two groups using the test and Dunnett’s T3method. It is considered to have statistics significance when P<0.05.Result 1.The expression of CD147mRNA detected by RT-PCR:There was mRNA in each group., the value of different groups of CD147mRNA also have significant differences (F=51.420, P=0.000).The expressions of CD147mRNA in blank control group and NC control group have no significant differences (P=0.796); Compared to control group, CD147mRNA were significantly reduced in shRNA lentivirus vector groups.(P=0.000). Among the lentiviral interference groups, A have the most significant effect on inhibiting the expressions of CD147mRNA expression, compared with blank control group reduced by57.72%(P=0.000).2. CD147protein of each group detect by ELISA:There was CD147protein in each group, the value of different groups of CD147protein also have significant differences (F=18.423, P=0.000).The expressions of CD147protein in blank control group and NC control group have no significantly differences (P=0.640); Compared to control group, the expressions of CD147protein were significantly reduced in shRNA lentivirus vector groups.(P=0.000, P=0.000, P=0.001, P=0.000). Among the lentiviral interference groups, A have the most significant effect on inhibiting the expressions of CD147expression, compared with blank control group reduced by42.40%%(P=0.000).3. MMP-2and MMP-9protein of each group detect by ELISA:There were MMP-2and MMP-9protein in each group, the value of different groups of MMP-2and MMP-9protein also have significant differences(F=206.012, P=0.000; F=9.385, P=0.001). Compared to control group, the expressions of MMP-2and MMP-9protein were significantly reduced in shRNA lentivirus vector groups.(P <0.05).Compared to blank control group and NC control group, MMP-2and MMP-9decreased to the lowest, the differences have statistically significant (P=0.014, P=0.003)4.The relativity of MMP-2, MMP-9and CD147in monocytes/macrophages The correlation coefficients of CD147and MMP-2, MMP-9in each group were r=0.899, r=0.814, both MMP-2and MMP-9have a significant positive correlation with CD147(P=0.000). Conclusion:1. The rabbit peripheral blood mononuclear cells can be successfully isolated by using a single nuclear cell separation liquid density gradient centrifugation and adsorptive method.2. We successfully designed and synthesized shRNA lentiviral vector for CD147gene, from which we selected specifically and efficiently block that can inhibit the expression of CD147shRNA.3. CD147regulates the expression of the matrix metalloproteinase, which can down regulate the expression of MMP-2, MMP-9. Part II The effect of extracellular matrix metalloproteinase inducer (CD147) shRNA lentiviral vector on the stability of atherosclerotic plaquesObject1. Establishing animal models of atherosclerotic plaque which are similar to human plaque by cold-induced endothelial injury with liquid nitrogen combined with high fat diet fed,in order to lay a foundtion for basic and intervention research in vulnerable plaques.2. Based on the self-invented rabbit models of AS rupture plaque, we screen lentiviral vector RNA that can effectively suppress CD147expression in rabbit peripheral blood monocytes/macrophage, locally infect with the rabbit carotid artery atherosclerotic plaques, to evaluate the effect of CD147RNAi in stabilizing the vulnerable plaques.Methods1. Establishing the animal model30healthy male New Zealand white rabbits were selected, they underwent cold-induced endothelial injury with liquid nitrogen in the right carotid arteries after3days adaptive feeding with regular chow, then were given a high-fat diet for ten weeks. 2Regional lentiviral transfection After completion of the preparatory procedures,2rabbits were randomly selected and sacrificed to check the plaque formation. According to the different viral infections, the rabbits were randomly divided into3groups and named respectively as following:①the blank control group (n=8) injected normal saline to the carotid plaque.②negative lentiviral control group (n=10) injected negative lentiviral vector to the carotid plaque.③LV-CD147interference group (n=10) injected CD147shRNA lentiviral vector to the carotid plaque.4weeks later, they were triggered with liquid nitrogen, then checked the changes of the plaques and observed the pathological morphology.3. Blood samples were collected for measuring total cholesterol (TC), triglycerides(TG) and low density lipoprotein(LDL)in serum prior to the experiment, on the10th weekend given high-fat diet and the4th weekend with viral interference, and48hours after triggering. The high-sensitivity Creactive protein (hs-CRP), matrix metalloproteinase-1(MMP-1), MMP-2, MMP-3and MMP-9were detected by enzyme-linked immunosorbent assay (ELISA) as well. tall rabbits were killed by an overdose of intravenous pentobarbital sodium48hours after triggering, the right carotid arteries were quickly removed for HE staining, Masson staining and Elastic tissue staining, and morphological characteristics of the plaque were observed under light microscopy. In order to identify the expression of inflammatory Factors and matrix metalloproteinase system in the plaque, monoclonal antibodies against NF-K,CD147,MMP-2,MMP-9and MMP-14were employed for immunohistochemical staining of the carotid arteries.4. Statistic analysis SPSS13.0was applied for description and analysis of the data. Measurement data was represented in the form of (mean±Std deviation) paired samples t-test was used to compare means of two paired samples. Multiple means’comparison adopted method of One-way ANOVA (Brown-Forsythe method was applied when equal variances not assumed). In cases those equal variances assumed, multiple comparisons of means adopted LSD test (Dunnett’sT3test was employed when equal variances not assumed).The significance level was0.05. Result1. The rabbits grew well generally. During the process of the test,5rabbits died,23rabbits were used in result analysis. Significant atherosclerotic plaques were observed in the lesion of right carotid artery after10weeks’high-fat diet. On the10th weekend, all the rabbit had significantly higher level of serum total cholesterol, triglycerides and lowerdensity lipoprotein than that prior to the experiment,(t value of each group was-42.117,-40.208,-30.293, P=0.000).2. Variation of serum CD147concentration:Prior to the experiment, on the10th weekend given high-fat diet and the4th weekend with viral interference, and48hours after triggering,CD147expression in serum has significant difference in the same group(F=66.651,P=0.000). CD147expression in serum has significant difference in each group at the same time (F=21.849, P=0.000). Time and interference factors have interaction effect (F=6.474,P=0.000).At4th weekend of lentivirus interference, CD147level of shRNA lentivirus group was significantly lower than other two negative groups(P=0.000).After48hours of liquid nitrogen activation, CD147level obviously elevated in3groups, there was no significant increase in CD147interference group comparing with control group (P=0.000).3. Variation of serum MMP-1concentration:Prior to the experiment, on the10th weekend given high-fat diet and the4th weekend with viral interference, and48hours after triggering, MMP-1expression in serum has significant difference at the same group(F=17.695,P=0.000). MMP-1expression in serum has significant difference in each group at the same time (F=21.849, P=0.000). Time and interference factors have interaction effect (F=7.407,P=0.000). CD147level of shRNA lentivirus group significantly increased at10th weekend given high-fat diet and48hours after triggering (P=0.000;P=0.012), MMP-1after lentiviral interference reduced significantly compared to10th weekend (P=0.002),there was no difference compared to48hours after triggering (P=0.314)4. Variation of serum MMP-2concentration Prior to the experiment, on the10th weekend given high-fat diet and the4th weekend with viral interference, and48 hours after triggering, MMP-2expression in serum has significant difference at the same group(F=15.233,P=0.000).Time and interference factors have interaction effect (F=7.971,P=0.000). MMP-2level of CD147shRNA lentivirus group significantly increased at10th weekend given high-fat diet and48hours after triggering (P=0.000;P=0.012), MMP-2after lentiviral interference reduced significantly compared to10th weekend (P=0.001),there was no difference compared to48hours after triggering (P=0.415).MMP-2expression of CD147shRNA lentivirus group after4weeks of lentiviral interference and48hours after triggering significantly decreased compared to the other two groups,which has a statistically significant (P=0.001, P=0.000)5. Variation of serum MMP-3concentration:Prior to the experiment, on the10th weekend given high-fat diet and the4th weekend with viral interference, and48hours after triggering, MMP-3expression in serum has significant difference at the same group(F=61.345,P=0.000). MMP-3expression in serum has significant difference in each group at the same time (F=8.901,P=0.000).Time and interference factors have interaction effect(F=8.202,P=0.000). MMP-3level of CD147shRNA lentivirus group significantly increased at10th weekend given high-fat diet (P=0.000); MMP-3after lentiviral interference has no significant decline compared to10th weekend (P=0.481),there was no difference compared to48hours after triggering (P=0.934).MMP-9expression of CD147shRNA lentivirus group after4weeks of lentiviral interference and48hours after triggering significantly decreased compared to the other two groups,which has a statistically significant (P=0.001, P=0.000).48hours after triggering, MMP-3has a significant difference among the three groups.(F=16.980, P=0.000); CD147shRNA lentiviral interference group significantly decreased compared to the other two groups,which has a statistically significant (P=0.000).6. Variation of serum MMP-9concentration:Prior to the experiment, on the10th weekend given high-fat diet and the4th weekend with viral interference, and48hours after triggering, MMP-9expression in serum has significant difference at the same group(F=28.399,P=0.000). MMP-9expression in serum has significant difference in each group at the same time (F=123.432,P=0.000).Time and interference factors have interaction effect (F=13.609,P=0.000). MMP-9level of CD147shRNA lentivirus group significantly increased at10th weekend given high-fat diet (P=0.000), MMP-9after lentiviral interference reduced significantly compared to10th weekend (P=0.004),there was no difference compared to48hours after triggering (P=0.661).MMP-9expression of CD147shRNA lentivirus group48hours after triggering significantly decreased compared to blank control groups,which has a statistically significant (P=0.000)7. Variation of serum hs-CRP concentration:Prior to the experiment, on the10th weekend given high-fat diet and the4th weekend with viral interference, and48hours after triggering, hs-CRP expression in serum has significant difference at the same group(F=1604.86,P=0.000). hs-CRP expression in serum has significant difference in each group at the same time (F=118.582,P=0.000).Time and interference factors have interaction effect (F=101.302,P=0.000). hs-CRP expression of CD147shRNA lentivirus group significantly increased at10th weekend given high-fat diet、4weeks of lentiviral interference and48hours after triggering (P=0.000), there was no difference after lentiviral interference and at10th weekend (P=0.096),48hours after triggering significantly increased, which has a statistically significant (P=0.000) hs-CRP expression of CD147shRNA lentivirus group after4weeks of lentiviral interference significantly decreased compared to the other two groups,which has a statistically significant (P=0.000)ConclusionOur research indicates that the series of matrix metal protease protein expression in CD147interfering group significantly reduced compared with blank control and negative control group after4weeks, and there was no obvious increase in expression by stimulation with li quid nitrogen for48h. Therefore, we assume that CD147shRNA lentiviral vector may play an important protective role in plaque stability of vulnerable plaque animal models by effectively silence CD147gene, and it could open a brand new way for the prevention and treatment of acute cardiovascular and cerebrovascular diseases. |
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