The Study Of Atherosclerosis And The Filter Of Different Expression Gene In Intermittent Hypoxia State

Backgroud:Obstructive sleep apnea/hypopea syndrome (OSAHS) is a kind of disease with latent hazard, which has complicated compact to human organism. Besides direct damage to respiratory system, OSAHS also can lead to disorder of cardiovascular system. Intermittent hypoxia (IH) is a specific type of hypoxia of OSAHS. The feature of IH is the repeated hypoxia with a interval of 20～120 seconds and PaO₂ return to the normal line in the interval. Present datas have showed that IH is different from persistence hypoxia and IH may startup a different intercellular signal pathway compared to persistence hypoxia. IH is easy to involve the cardiovascular system and induce multiple high risk complication. Therefore, IH is the independent risk factor of cardiovascular system disorder. OSAHS is closely related to atherosclerosis (AS) but it is unclear now if IH can influence the development of AS. Objective:To construct the animal model of AS development by simulating intermittent hypoxia situation of human OSAHS. To filter the different expression genes by cDNA micro array and construct suppression subtractive hybridization cDNA library of rabbit liver in intermittent hypoxia with hyperlipemia. Research the change of related gene of liver in the development of AS on hyperlipemia status. Explore the potential molecular mechanisms of AS development on IH status and offer the theory basic for the treatment of AS.Methods:Selects 24 health experimental rabbits, female, the male gender is half and half, 2～4 months old. The 24 experimental rabbits were divided into four groups according to the foundation feed, high fat diet (HFD) and the intermittent low oxygen intervention condition. six experimental rabbits of IH group(foundation feed + intermittence low oxygen),six experimental rabbits of HFD group (high fat feed + intermittence low ox),six experimental rabbits of HFD+IH group (high fat feed + intermittence low oxygen),six experimental rabbits of C group(foundation feed raising take comparison). After one week of the compatibility feeds the standard foundation , we weight the experimental rabbit, take the empty stomach blood 2ml from each rabbit ear reason vein to determine blood serum total cholesterol (TC), triglyceride (TG), the low density lipoprotein - cholesterol (LDL-C). Put the IH group and the HFD+IH group in the intermittent low oxygen cabin during making animal mold experiment period every day at 9:00Am～5:00PM,we use oxygen meter to monitor the oxygen concentration of intermittence low oxygen cabin in order to control air transfusion and air bleed. Sufficiently enters the mixture of nitrogen and air(90% N₂ and 10% O₂)into the intermittent low oxygen cabin, under the electric circuit control respectively by the 2L/min speed of flow input, each time circulates 8 minutes, meaning 4 minutes sufficiently enter the nitrogen, in the maintenance of the oxygen lowest density reach 8.5%, the maintenance time is 1 minute, afterwards 2 minutes discharge the mixed air in the cabin, makes the oxygen concentration in the cabin restore to 21% and maintains 1 minute, to next circulation, according to this condition control intermittence low oxygen condition. Every day except the experimental group, rabbits living conditions of C group, the IH group, the HFD group, HFD + the IH group is same. After 12 weeks, executes the experimental rabbits, from the aorta outset department to the abdomen aorta fraction place entire journey dissection exposition, observes the atherosclerosis situation, separately cut each group of experimental rabbit aorta, the formaldehyde fixedly makes the paraffin section, uses in HE dyeing and Sudanese III dyeing. Quickly freeze other parts and store in nitrogen canister of -70℃super cold refrigerator spare. At the same time, observes the histo-pathology change of abdomen aorta, heart, liver, lung and so on.Extract the abdomen aorta's total RNA of HFD group, the HFD+IH group and the IH group experimental rabbit, reverse transcription to prepare hybridization probe, hybrid with American Human Cardiovascular Disease Gene Array II (HS-038) chip of Affymetrix Corporation. After scanning the hybridization signal with the ScanArray4000S , we use the Microarray Suite Version 5.0 software to analyze. Uses the RT-PCR method to confirm partial differential expression gene.Extract liver mRNA of HFD group and HFD+IH group, nidus PCR was used to synthesize double cDNA, purified dscDNA, Rsal enzyme-cutting dscDNA, and conjugated PCR production and T/A carrier, Inverting Bacillus coli Lib to carry out amplification. Culling masculine clone to run PCR amplification evaluation. Reciping masculine clone to extract Plasmid as formwork to run PCR amplification evaluation. After two turns of differential hybridization and rejection capability PCR, we obtain the differential expression cDNA of the two. Using cDNA of PFD group and PFD+IH group tagged byα-32^p as exploring probe, to running Northern Dot Blot reverse hybridization with Hybond membrane prepared by masculine clone PCR product , radioactivity developing, Hybridization signal was scanned by Scan Array, and analyzed by Scan Analyze soft ware. We marked the clone which Hybridization signal has double gaps. We picked twelve visible different clone to run sequencing. And the results run homolog matching analysis with NCBI database. Using fluorescent quantization PCR to validate rabbits' liver correlated differential expression genes.Results:1. Changes of experimental rabbit's weight under the IH condition. We weight all the groups of experimental rabbit after a week of adaptability feed, The weight of HFD group is 2.201±0.226 kg, The weight of C group is 2.102±0.234 kg, The weight of IH group is 2.301±0.347 kg, The weight of HFD+IH group is 2.212±0.126 kg, There is no statistic significance in Group comparison difference, The result means body weight of experimental rabbit selected in this experiment has the relative homogeneity, which established the same reference for the following establishment animal model experiment. After gives the intermittent low oxygen intervention factor, we observe the weight change of rabbit by every 6 weeks. There is no statistic significance (p>0.05)in the comparison of rabbit's weight differences between The HFD group, the HFD+IH group, the IH group and C group. At the 12th week of experimental each group of experimental rabbit's body weight increases comparing to the 6th week. The rabbit's body weights of HFD group and the HFD+IH group are higher than the IH group and C group of experimental rabbits, but the difference has no statistic significance (p > 0.05), The IH condition does not have the influence regarding the experimental rabbit body weight.2. Change of experimental rabbit's blood fats. The difference of groups has no statistic significance (p>0.05) before experiment. with the increase of high fat feed and the prolong of feed time, TC, TG and the LDL-C level of HFD group and HFD+IH group is higher than C group, the difference has statistic significance (p < 0.05). TC, TG and the LDL-C level of HFD group and HFD+IH group is higher than the IH group, the difference has statistics significance (p < 0.05). But the difference between C group and IH group has no statistic significance (p > 0.05).3. The Experimental rabbit of IH group's most low point artery blood oxygen partial pressure is 20.9mmHg～29.7mmHg in the intermittent low oxygen cabin, the lowest blood oxygen degree of saturation scope is 31.2%～58.3%, After restoring the oxygen concentration to 21% in the cabin, the artery blood oxygen partial pressure is 71.6mmHg～106.4mmHg, the artery blood oxygen degree of saturation is 92.2%～ 97.4%, conforms to the diagnosis standard of human blood oxygen degree of saturation (SaO2 < 80%).4. Under the IH condition Observing roughly the abdomen aorta specimen, the formation of AS exist difference. The abdomen aorta of C group experimental rabbit is soft; the internal membrane is smooth and have not atherosclerotic plaque and the formation of lipin mottling. Only one example of IH group has the 3mm long fat grain in the abdomen aorta, The pathological changes of HFD group and HFD+IH group are suffusion with naked eye, involve the hole abdomen aorta, Mostly assumes the strip rope massive, continually Large expanse of distributes, color is yellowish gray, obviously highlights to the aorta internal membrane surface. The vessel wall is thickening and stiff, endhymenine has the yellow and white fatty streak and eminence fatty stripe, which is rope shaped and the irregular small laminated distributed. the experimental rabbit under high fatty feed condition is easier to have the AS pathological change.5. After dyeing the abdomen aorta tissue under IH condition with HE, the structure existence obvious difference observed with microscope. The endomemembrane of C group aorta is smooth and can not find froth cell under the endomemembrane, tunica media, and involucrum are clearly discernible, each structure is normal, non- lipin deposition. The vessel internal membrane of IH group is obviously discontinuity disorganization. The endothelial cell is atomic and absence and smooth muscle cell reduce can be found obviously in HFD group and the HFD+IH group abdomen aorta with light microscope. In the multilayered froth cell tightly pastes the tension board to pile up, the partial packing to the intermediate deck froth cell, under microscope many froths cell gather under endothelia, analogue of lipin drifts away to the extracellular, form athermanous plaque. Specimen of IH group and HFD+IH group has obvious difference under microscope. This result showed AS is easy formed under the IH condition.6. Under the IH condition heart, liver and lung and so on are easily involve in damage. C group rabbit cardiac muscle organizational structure is normal, There is inflammation cells infiltrating in not interstitial substance. Cardiac muscle cell of HFD group, IH group and HFD+IH group is swelling, endochylema assumes the pellet condensation and the distribution is non-uniformity. Interstitial edema, inflammatory cell infiltrate, interstitial fibrosis, cardiac muscle fibers arrangement disorder, cardiac muscle cellular edema. Lungs organizational structure of C group is clear, lung arteriole vessel wall is thin, The HFD group, and the IH group and the HFD + IH group rabbit lung tissue have obviously the inflammation cell infiltrating. Transaction cell population of lung arteriole increases, vessel wall of lung arterio generally thicken, lumens obviously become narrow.7. Comparison of the thickness of the tunica intimae and media of abdominal aorta between each group. The thickness of the abdominal aortic tunica intimae from group C, group IH, group HFD and group HFD+IH are (5.57±1.15)μm, (32.6±7.91)μm, (43.6±2.67)μm and ( 76.4±12.32 )μm, respectively. Comparison of group IH, group HFD and group HFD with group IH showed statistically significant difference (p<0.05); group IH and group HFD showed statistically significant difference compared with group HFD+IH (p<0.05), comparison between group IH and group HFD showed no statistically significant difference (p>0.05). The ratio between the abdominal aortic tunica media and tunica media from group C, group IH, group HFD and group HFD+IH are (4.57±1.23) %, (25.4±1.21) %, (29.2±1.35) %, (76.4±1.03) %; Comparison of group IH, group HFD and group HFD with group EH showed statistically significant difference (p<0.05); group EH and group HFD showed statistically significant difference compared with group HFD+IH (p<0.05), comparison between group IH and group HFD showed no statistically significant difference (p>0.05).8.The percentage of all group rabbits abdominal aortic fatty steaks lipid plaque area compared with the area of vascular .The results in C group,IH group,HFD group,HFD+IH group showed that the percentage: Group C (0.51±0.14%), IH (1.25±0.32%), the HFD group ( 42.37±12.45)%, + EH HFD group (74.36±11.36)%. IH group, the HFD group and the group with HFD IH + C group, the difference was statistically significant (p <0.05); HFD + IH group and IH group, the HFD group, the difference was statistically significant (p <0.05); But the difference was not statistically significant (p> 0.05), between HFD group and the C group. 9. In the first gene-array, compared HFD+IH group with HFD group, 131 genes were up-regulated, 93 genes were down-regulated. In the second gene-array, compared HFD+IH group with IH group, 56 genes were up-regulated, 52 genes were down-regulated. To classify with cell signaling,immune inflammatory reaction,ecto-matrix degradations blood clotting and fibrinolysis,apoptosis,oncogene or anti-oncngene,correlated SMC and cell movement, 46 genes were selected as differentially expressed genes, including 29 up-regulated and 17 down-regulated genes. Parallel compared the first gene-array and the second, 20 genes were selected, and the difference was more than 2 times, including 3 apoptosis related genes,5 cell signaling related genes,6 immune genes,2 cytoskeleton and cell movement genes,3 metabolism genes and 1 protein synthesis gene; 17 down-regulated genes were selected, including 2 cell signaling related genes,1 immune inflammatory reaction genes,4 ecto-matrix degradation genes,2 blood clotting and fibrinolysis genes,3 apoptosis related genes,2 correlated SMC genes,1 oncogene or anti-oncngene,2 cell movement genes. those results showed that many genes take part in the formation of AS in the condition of IH10.Based on the results of the gene-array, we validate the different expression of inflammation- related gene IL-8,COX-2 in abdominal aorta tissues along the group of C,IH,HFD and HFD+IH by RT-PCR. The results revealed that abdominal aorta tissues in the HFD+IH and HFD group revealed significantly higher average levels of IL-8 mRNA than the C and IH group (mean±SD 2.43±0.41 vs 1.97±0.23 vsl.27±0.11 vs 0.47±0.17 respectively, and as well as the levels of COX2 mRNA (mean±SD 2.37±0.59 vs 1.79±0.21 vsl.35±0.07 vs 0.57±0.09 respectively) ,the differences have statistical significance (p<0.05) .With an Analysis of the Relevance between the expression levels of IL-8 mRNA in IH and HFD group and the pathology change in abdominal aorta tunica intima, we discovered:â‘ in the abdominal aorta tunica intima of IH group these is significant relevance along the expression levels of IL-8 mRNA with the area of plaque,the thickness of tunica intima,the ratio of thickness between tunica intima and tunica media( 0.903,0.875,0.896, p< 0.05).â‘¡In the abdominal aorta tunica intima of HFD group these is significant relevance along the expression levels of IL-8 mRNA with the serum LDL-C,the area of plaque,the thickness of tunica intima,the ratio of thickness between tunica intima and tunica media (0.923,0.814,0.821,0.877, p< 0.05) .With an Analysis of the Relevance between the expression levels of COX-2 mRNA in IH and HFD group and the pathology change in abdominal aorta tunica intima, we discovered:â‘ in the abdominal aorta tunica intima of IH group these is significant relevance along the expression levels of COX-2 mRNA with the area of plaque,the thickness of tunica intima,the ratio of thickness between tunica intima and tunica media ( 0.927,0.867,0.853, p< 0.05).â‘¡In the abdominal aorta tunica intima of HFD group these is significant relevance along the expression levels of IL-8 mRNA with the serum LDL-C,the area of plaque,the thickness of tunica intima,the ratio of thickness between tunica intima and tunica media (0.852,0.945,0.875,0.896, P< 0.05). Those results showed that the main development mechanism of AS maybe is the inflammatory reaction which is mediated by IH .11. Second round suppressive PCR product were cloned into pMD19-T vector, transformed into E.coli, Randomly picked 300 white clones, positiveness confirmed by PCR amplification using the inside sequences of ligatorl and 2R as the primers.325 and 244 reverse positive clones were obtained by using forward and reverse SSH, respectively, the positiveness is 96%.The inserting fragments varied from 100 to 600bp in size, which meant the inserting fragments of plasmid are differ from each other.12. We picked 12 different clones, after sequenced the clones and BLAST the results in the NCBI database, we got two known genes (PC4,COX2,MYL6,HDAC7), two sequences which homologous to the genome contigs and the new sequence not homologous to any known genes. Among the sequencing result of total 12 clones, two clones got identical output, one got nothing. The two sequences which are homologous to the genome contigs can be covered by multiple ESTs from human EST database, indicating HDAC7 is probably a new gene.Conclusion:1. In this experiment, we successfully build the rabbit model of promoting the atherosclerosis (AS) formation under the state of intermittent hypoxia (IH) by using intermittent hypoxia cabin. IH is an new important risk factor of promoting the AS formation.2. Multiple genes' alteration are involved in the formation and progression of AS under the state of IH. Differential expression profiling may provide a new tool to reveal the mechanism of atherosclerosis.3. Under the state of IH, the local inflammation of the blood vessel wall in conjunction with hypercoagulable state of blood and the deposition of fat particle may lead local atherosclerosis mass formation. Related regulatory genes in the intermittent hypoxia induced matrix degradation play a role in the degradation of fibrocollagen and the extracellular MMP disassembling. One of its probably regulating methods is that intermittent hypoxia may lead to the abnormality of lysine oxydase and pro-fibrin which are essential in the maturation of elastin, then result in the polymerization of elastin to elastin fibra prevented by poly-glycoprotein in sclerotic artery wall, and IL-8 and COX-2 play an important role in this process.4. The inflammatory factors produced during the stress and inflammatory reaction of liver under IH may also enter the blood circulation, involved in the inflammatory reaction of blood vessels. The overexpression of HDAC7 gene in hepacytes is a novel mechanism of promoting the formation and progression of atherosclerosis.