| BACKGROUNDAcute lung injury ?ALI? is caused by a variety of non cardiogenic factors of progressive dyspnea, refractory hypoxemia and pulmonary edema, pathological feature of the alveolar capillary barrier injury, increased permeability, ventilation/ perfusion imbalance caused by respiratory failure, the evolution of acute respiratory distress syndrome ?ARDS?, lipopolysaccharide ?LPS? and direct injury of microvascular endothelial and indirect effects play an important role, is involved in many inflammatory cells and cytokines, a large number of polymorphonuclear neutrophils, macrophages and other inflammatory cell infiltration and activation and release of inflammatory cytokines. Although the symptoms of ALI/ARDS is improved, much research progress on the control of mortality of drugs, but the mortality is still as high as 40-60%, because of this, to find new effective treatment strategies The repair of ALI/ARDS damage is still an urgent problem to be solved.Lipopolysaccharide ?LPS?, also called endotoxin can be induced by endotoxemia, resulting in septic shock, leading to multiple organ damage and failure, but the heart is the most commonly involved organs during endotoxemia, but the mechanism of injury and cardiac function in endotoxemia myocardial insufficiency is not very clear. Inflammation and apoptosis is an important feature of septic shock, septic shock in all, about 40%of the patients are often accompanied by varying degrees of cardiac insufficiency. Although the bacteria can be the immune system or effective antibiotics to kill, the shock does not necessarily improve.LPS release into the blood. Can induce macrophages, dendritic cells release TNF-alpha, IL-lbeta and other inflammatory factors, and induce cell apoptosis.Mesenchymal stem cells ?MSCs? is a kind of cell potential to the lack of specific cell markers, body characteristics of MSCs or MSCs like cells they may originate from bone marrow, fat, umbilical cord blood, placenta, skeletal muscle and tendon, bone marrow mesenchymal stem cells ?BM-MSCs? have multilineage differentiation, immune regulation and paracrine characteristics in wound healing, tissue regeneration and functional reconstruction, play a positive role on immune regulation and treatment, BM-MSCs can inhibit the apoptosis of anti-inflammatory and immune regulation, reducing acute lung injury ?ALI? induced by endotoxin, improve the lung function. However, compared with BM-MSCs, umbilical cord mesenchymal stem cells ?UC-MSCs? are easy to extract, large amount of cells, the proliferation rate is high.Angiopoietin ?Ang? is a group of endothelial cells secreting specific type of growth factor in angiogenesis, embryonic development and wound healing plays an important role in the expression of Angl in different cells, including perivascular cells, smooth muscle cells, fibroblast cells, megakaryocytes, can reduce the inflammatory response, inhibit cell apoptosis and decreased vascular permeability, maintain alveolar capillary membrane stability and play a role in ALI, Angl modified BM-MSCs can play a synergistic role with MSCs in anti inflammation and stable intima, play a stronger role to reduce blood vessel leakage. But there are relevant reports not only treatment of ALI but also intervention myocardial injury and heart function of UC-MSCs modified by Angl gene. Therefore, this study takes UC-MSCs as the carrier, to carry and high expression of Angl gene by gene transfection technology, enhance the role of the UC-MSCs, as the experimental base for further ALI treatment provides the basis. UC-MSCs-Angl inhibit the inflammatory response and myocardial injury of myocardial cell apoptosis induced by LPS; The influence of UC-MSCs-Angl on LPS induced myocardial after the injury of cardiac structure and function, to provide the experimental basis for the septic shock in the myocardial injury of cell and gene therapy.OBJECTIVE1. The separation, cultivation and identification of human UC-MSCs; Building lentiviral vector carrying GFP and Angl gene.The expression of GFP or Angl was detected using a fluorescent microscope and western blotting.2. To establishment the experimental method of Angl gene modified UC-MSCs in the treatment of LPS induced ALI. To study the improvement effect by injecting Angl gene modified UC-MSCs on LPS induced ALI on pulmonary edema, systemic inflammatory response, cell implantation rate, survival rate and so on.3. To investigate the possible paracrine and immune modulation mechanism of Angl gene modified UC-MSCs in the treatment of ALI, and to study whether or not to promote the survival of UC-MSCs in injured lung tissue.4. Observation of UC-MSCs-Angl inhibited LPS induced myocardial injury and inflammatory reaction and apoptosis of cardiomyocytes.5. To study the effect of UC-MSCs-Angl on cardiac structure and function after LPS induced myocardial injury, and to provide a theoretical basis for its application in cardiovascular diseases.MATERIALS AND METHODS1.The biological characteristics and angiogenin-1 genetic modification of human umbilical cord mesenchymal stem cells1. Generation and administration of UC-MSCs and cell surface antigen phenotyping detectionThe sterile umbilical cords were cut into small pieces and placed in plates with low-glucose Dulbecco-modified. Eagle medium ?L-DMEM? supplemented with 10% fetal bovine serum. The adherent fibroblast-like cells were cultured to confluence,and the cells were used after five or more passage.2. The general characteristics of UC-MSCs were identified through morphologi cal observation and flow cytometryFifth-passage cells were collected and then stained with either fluorescein isothio-cyanate-conjugated or phycoerythrin-conjugated monoclonal antibodies in 100 ?l phosphate buffers for 20 min at room temperature.The antibodies used were against human antigens CD29, CD34, CD44, CD45, CD31, CD90, CD105, and CD133. Positive cells were counted and compared to the signal of corresponding immunoglobulin isotypes.3. Identification induced differentiation capacity of UC-MSCsUsing chemical induction method, these cells were mduced into osteoblasts and adipocytes. Functional differentiations were evaluated using cytochemical stains.4. Lentiviral vectors construction and lentivirus infection4.1 The angiogeninl mRNA sequence was searched in the U.S. National library of medicine of the National Center for Biotechnology Information ?NCBI? nucleotide database, designed of both ends with 1 and Sal BamH I enzyme sites primers. The total RNA was extracted from 100mg normal human umbilical cord by Trizol reagents. The Ang1 cDNA fragment was amplified by RT-PCR.4.2 Lentiviral vectors containing green fluorescence protein ?GFP? were employed in order to achieve high efficiency of introduction and subsequent stable expression of Angl in MSCs. Recombined pGC-LV-GV287-GFP vector with the Angl ?NM001146? gene ?LV-Angl? and pGC-LV-GV287-GFP with a scrambled control sequence ?LV-GV287? were constructed. MSCs were then infected with the above lentiviral vectors. A total of 1×105cells/ml MSCs cells were seeded in a six-well cell plate and further incubated for 24 hours to reach 40% confluent, and then infected with LV-Ang1?Ang1 overexpression group?, LV-GV287 ?negative control group? and no infection ?non-transfected control group? by replacing the infection medium containing recombinant vectors at a multiplicity of infection ?MOI? of 8 plaque-forming units per cell.Plates were then incubated for 24 hours prior to having their media changed to fresh, virus-free media.72 hours later, the GFP density contained by lentivirus was detected to evaluate the efficiency of infection using fluorescent microscopy, and cells were harvested for Western Blot analysis.5. Statistical AnalysisThe data were analysed by SPSS 17. The numerical variable data in mean standard deviation of that between the two groups were compared using small sample t test, and multiple groups were compared using One-Way ANOVA to P<0.05 for the analysis of variance, the difference was statistically significant.II.Human umbilical cord mesenchymal stem cells carrying angiopoietin-1 alleviated LPS-induced acute lung injury in rats1. Rat ALI model making:125 SD rats were selected and randomly divided into five groups:control group. LPS group, Fibroblast group, MSCs group and MSCs-Ang1 group ?n=25 for each group?. Rats in the latter four groups were intraperitoneal inj ected with 10mg/kg?body weight?of LPS to produce a sepsis ALI model.2. UC-MSCs infusion treatment of lung injury:After LPS injection induced lung injury, saline containing 5×105 UC-MSCs in 300ml were injected through tail vein in MSCs group. Total 300ml saline containing 5×105 fibroblasts?MRC-5 cell line? were injected in Fibroblast group.Total 300ml saline containing 5×105 MSCs-Ang1 were injected in MSCs-Angl group, while the other two groups were injected with nomal saline volume. Each group were sacrificed 3-5 rats at 6 hours,24 hours, 48hours,8 days and 15 days after infusion therapy respectively. The plasma and lung tissue were collected for routine biopsy, bronchoalveolar lavage fluid neutrophil count, lung wet-to-dry ratio and serum inflammatory cytokines TNF-?, TGF-?1, IL-10, and IL-6 ELISA analysis.3. To detect the migration and implantation of MSCs-Angl in lung tissue:By fluorescence microscopy GFP positive cells were observe to assess the distribution and the rate of implantation of exogenous MSCs-Angl in lung tissue. Twenty fields were randomlyselected at x40 magnification and the GFP-positive and GFP-negative cells were counted.Real time quantitative RT-PCR was used to detect of exogenous GFPmRNA expression in receptor lung tissue.4. Survival analysis:Five groups healthy SD rats ?n= 10? were used for survival experiments, record the survival days of animals, observe the living conditions of animals during the experiment, calculate the survival rate and draw the survival curve.5. Statistical Analysis:The data were analysed by SPSS 17.?. Human umbilical cord mesenchymal stem cells carrying angiopoietin-1 on LPS-induced left ventricular myocardial remodeling and function in rats.1. ECGAccording to the different time points of the experiment, the electrocardiogram ?I, ?, ?, aVR, aVL, aVF? ECG of limb lead was described, and the cardiac electrical activity of rats in each group was observed and ECG was recorded.2. EchocardiographyEchocardiography was performed at different time points. The indexes were as follows:left ventricular end diastolic diameter ?LVIDs?, left ventricular end diastolic diameter ?LVIDd?, ejection fraction ?LVEF?, left ventricular fractional shortening ?FS?.3. Millar catheter measurement of left ventricular functionBefore the rats were sacrificed after anesthesia exposure of left ventricular apex, with 10ml needles, a Milllar catheter electrode probe directly for precise control of apical puncture into the left ventricle, the analysis software of cardiovascular parameters, left ventricular systolic pressure ?LVSP?, left ventricular end diastolic pressure ?LVEDP?, maximal rate of left ventricular pressure ?+dp/dt?, left ventricular pressure decline rate ?-dp/dt?.4. Serological markers detection6h,24h,48h,8d,3-5 rats in each group were sacrificed respectively. The serum was collected, and the serum level of\cTnI?NT-proBNP were measured by immunoassay.5. Cardiac tissue pathology detectionAfter the animals were sacrificed, cardiac tissue were fixed, dehydrated, transparent, soaked in wax, paraffin embedded, sliced, conventional HE staining,3% glutaraldehyde fixed myocardial tissue after the transmission electron microscope observation.The paraffin sections were stained by immunohistochemical method to detect apoptotic cells under the microscope, and statistical analysis were used to detect the left ventricular myocardium.6. Real-time quantitative RT-PCR gene level detectionTrizol method to extract total RNA, RT-PCR, TNF-a, IL-6, RIP3 and mRNA Caspase-3 expression levels of. RT-PCR detection of mRNA GFP expression of exogenous MSCs-Angl cells in the cardiac tissue of the implant.7. Western blotExtracting total protein, Western-Blot, TNF-a, IL-6, apoptosis related RIP3, Caspase-3, Cleaved Caspase-3 protein expression in the left ventricular myocardium.8. Statistical AnalysisThe data were analysed by SPSS 17. The numerical variable data in mean standard deviation of that between the two groups were compared using small sample t test, and multiple groups were compared using One-Way ANOVA to P<0.05 for the analysis of variance, the difference was statistically significant.RESULTSI.The biological characteristics and angiogenin-1 genetic modification of human umbilical cord mesenchymal stem cells1. MSCs derived from umbilical cord can be exuberant proliferated with typical fibroblast cell morphology. They can express typical MSCs cell surface antigen such as CD29, CD44, CD90 and CD105. Nonhematopoietic or endothelial cell markers such as CD31, CD34, CD133 and CD45 couldn't be detected. Osteoblasts and adipocytes differentiation could be achieved after induction with the expression of specific markers respectively.2. Infection efficiency of lentivirus vectors.In order to achieve high efficiency of introduction and subsequent stable expression of Angl in MSCs, we tried to import this gene by infecting MSCs with Ang1 lentiviral vectors containing GFP. The recombinant lentivirus vector LV-Ang1 was successfully constructed and infected MSCs. The stably infected MSCs expressed GFP after infection by the lentiviral vectors at different MOIs.3. GFP expression was detected sixty hours after infection using fluorescence microscopy.The efficiency of the infection ?averaged proportion of GFP-expressing cells on the total cell count? was approximately 95% at an MOI of 8. GFP positive cells rate was 97.4% by flow cytometry instrument detection Consequently, an MOI of 8 was chosen for the next steps of this study.Furthermore, the effectiveness of the lentiviral infection of LV-Ang1 was also confirmed by Western blotting analysis. The protein expression of Angl in MSCs was significantly higher in the Ang1 overexpression group than that in the control groups.?.Human umbilical cord mesenchymal stem cells carrying angiopoietin-1 alleviated LPS-induced acute lung injury in rats1. Histopathologic examination and scoringSix hours after intraperi-toneal injection of LPS, the lung tissue capillaries expanded and became congested by a significant increase in neutrophils. At the same time, some small abscesses and bullae of lung also were found.These events peaked at the 48 hour time-point. MSCs-Ang1 rats also displayed moderate injury, but the severity was significantly less compared to the MSCs group after 24 hours of intraperitoneal injection LPS. The lung injury score was significantly lower in the MSCs-Ang1 group at 6hours,24hours,48hours,8days and 15 days time-points. In contrast rats given injection of human fibroblasts, had no improvement in lung injury.2. Neutrophil infiltration in the lungsThe total inflammatory cells counts in the BALF were increased mainly attributable to an increase in neutrophils at 6 hours,24 hours,48 hours,8days and 15days following administration of LPS.Treatment with MSCs-Ang1 further reduced the BAL neutrophils counts at 24hours and 48hours and neutrophils were close to a level similar to normal group at 8days and 15days.LPS also caused significant increase of MPO activity in the lung tissue after acute lung injury. These increases were reduced in MSCs-Angl group significantly.3. Lung wet-dry ratioLPS challenge produced significant lung oedema in the LPS group, as shown by the increased lung wet-dry ratio. MSCs-Angl treatment significantly attenuated the increase in lung wet-dry ratio compared with the MSCs group at 48hours and 8days after LPS inhalation.4. MSCs-Angl treatment attenuates systemic inflammation associated with LPSThe pro-inflammatory cytokine TNF-?, IL-6,TGF-?1 and anti-inflammatory IL-10 were detected in serum by ELISA. In the MSCs-Angl treated group, the LPS-induced increase of TNF-?,IL-6, TGF-?1 were significantly reduced, but not for the other three groups. In addition, the response of antiinflammatory IL-10 of MSCs-Angl and MSCs groups reached the peak at 48 hours after injection of LPS, and decreased gradually at the following time points.5. Engraftment of MSCs-Angl in lung tissuesUntil 8 days after injury, the GFP+cells quantity reached its peak.The average percentage of GFP+ cells was approximately 21% in the randomly selected fields for the MSCs-Angl group, compared with 10% in the MSCs group, suggesting that Angl may enhance MSCs recruitment and engraftment to the lung. In addition, the numbers of GFP+ cells in the severely injured areas were much higher than those in mildly injured areas. Quantitative RT-PCR detected of exogenous GFP mRNA expression of MSCs-Angl in receptor lung tissue.It indicated that reconstitution with GFP+ stem cells were successful.6. Survival analysisThere was a significant improvement in the survival rates between MSCs and MSCs-Angl groups.The 15d survival rate of the MSCs group was 50%?5/10? while that of the MSCs-Angl group reached 70%?7/10?. The differences among various groups were significant. Treatment with MSCs-Angl engraftment significantly decreased the rats'mortality after ALI.?. Human umbilical cord mesenchymal stem cells carrying angiopoietin-1 on LPS-induced left ventricular myocardial remodeling and function in rats.1. The electrocardiogramThe control group ECG QRS wave amplitude, arrhythmia in. LPS group with abnormal ECG findings, visible QRS wave alternans, tachycardia, bradycardia and other complex arrhythmia; MSCs and MSCs-Angl intervention, no obvious complex arrhythmia decreased, suggesting that MSCs-Angl partially improved the ECG activities.2. EchocardiographyChanges in the structure of left heart. Left heart chamber size, at the beginning of the experiment, LVIDs and LVIDd were no significant differences between groups ?P> 0.05?; 15 d experiment, compared with control group, LPS group LVIDd expand ?P< 0.05?;Compared with LPS group, MSCs with MSCs-LVIDd Angl intervention group were decreased, MSCs-Ang1 intervention group decreased ?P< 0.05?;Left ventricular systolic function. At the beginning of the experiment, each rat between the change of the FS and LVEF were no significant differences ?P> 0.05?.15 d experiment, LPS group compared with control group, FS, reduced LVEF were different degree ?P< 0.01-0.05?; Compared with LPS group, MSCs with MSCs-FS, LVEF Angl intervention group were different degree rise, MSCs-Angl group obviously was improved ?P< 0.01-0.05?.3. Millar catheter evaluated left heart function.At the end of the experiment, compared with control group, LPS group significantly decreased ?P< 0.01?, elevated LVEDP ?P< 0.01?,+dp/dt Max absolute value lower ?P< 0.01-0.05?,48 h-8 d hours were significantly;Compared with LPS group, MSCs with MSCs-Angl two groups after the intervention, LVSP increased obviously after MSCs-Angel intervention ?P< 0.05?,+dp/dt Max ?mmHg/s? were increased ?P< 0.05?.Compared with LPS group, two different LVEDP intervention group were decreased ?P< 0.01-0.05?, dp/dt Max ?mmHg/s? absolute value increased ?P< 0.05?. Prompt MSCs-Angl to left ventricular systolic function improved significantly after intervention.4. Detection of serology indexes.Compared with control group,24 h LPS group of cTnI levels began to rise and there are differences, cTnI levels peaked when 48 h,48 h-8 d continue to rise, prompt myocardial injury is heavier, but MSCs with MSCs-Angl intervention after LPS group there were significant differences ?P< 0.05?,8 d cTnl levels earlier to reduce, 15d reduce obviously, myocardial injury improved markedly.5. LPS induce left ventricular remodeling in rats myocardial inflammation, apoptosis, and the effects of MSCs-Angl transplantation5.1 LPS induced rat myocardial inflammatory reaction and the effects of MSCs-Angl transplantationHE staining. LPS group compared with normal control group, visible focal interstitial edema, myocardial fiber rupture, clutter, interstitial vascular congestion, local granulocyte increased;Compared with LPS group, MSCs with MSCs-Ang1 two groups after the intervention, pathological conditions improve.The expression of myocardial inflammatory factor TNF alpha and IL-6 after MSCs-Angl transplantation.Immunohistochemical detection of the expression of TNF alpha and IL-6:the control group, small, thin light particles in the cell;LPS group of cytoplasm see more dark brown granules, MSCs with MSCs-Angl compared two groups after the intervention and LPS group, see brown scattered and dilute particle distribution, thin, reduce.Western Blot detection TNF-alpha, the expression of IL-6 protein:LPS group of TNF alpha, IL-6 expression significantly higher ?P< 0.01?.After MSCs with MSCs-Angl intervention, compared with LPS group, both myocardial tissue TNF alpha, IL-6 protein expression in different degrees of decline ?P< 0.05?.5.2 LPS induced rat myocardial tissue cell apoptosis and the effects of MSCs-Angl transplantationElectron microscope apoptosis cells. LPS group showed mitochondrial disorders, significantly increased, swelling, and some mitochondria dissolved, crest fracture, some muscle wire fracture; MSCs with MSCs-Angl after the intervention, compared with the LPS group, various lesions are mitigated, MSCs-Angl intervention is more improved than MSCs.TUNEL staining observation. The control group, LPS group left ventricular apoptotic cells were significantly increased ?P< 0.01?. MSCs-Angl group after the intervention compared with LPS group, the apoptosis rate decreased significantly ?P< 0.01?.MSCs inhibit expression of myocardial apoptosis related RIP3.Immunohistochemical staining. The control group, LPS group RIP3 tan increase positive particles expression, and distributed around the nucleus is more, MSCs with MSCs-Angl compared with LPS group, two groups of tan positive particles in the cell expression is reduced, and after MSCs-Angl injection, tan particles decreased significantly.Western Blot detection. The RIP3 protein expression levels of LPS group were significantly higher than intervention groups?P<0.01?; But after cell transplanted the RIP3 protein of MSCs and MSCs-Angl groups, were differented from LPS group ?P < 0.01-0.05?. MSCs inhibited expression of myocardial apoptosis related Caspase 3.Western Blot -detection Cleaved Caspase 3/Caspase 3 protein expression ratio. Compared with control group, LPS group ratio significantly increased ?P< 0.01?;But after MSCs and MSCs-Angl intervention, compared with LPS group, the ratio of treatment groups were differently decreased ?P< 0.01-0.05?.6. The GFP mRNA express exogenous MSCs-Angl cells in myocardial tissue implant testAt 48 h after transplantation group,8 d MSCs Angl GPF mRNA expression was significantly higher than the no-load MSCs, was statistically significant ?P< 0.05?;In the early MSCs-Angl group GPFmRNA expression is higher than the no-load MSCs but without statistical significance.24 h,48 h after transplantation,8 d, three groups of cell rt-pcr detection GPF mRNA expression increased ?P< 0.05?, suggested that Angl could promote MSCs in the survival and the myocardial tissue implantation.CONCLUSIONS1. Human umbilical cord can be separated in a large number of stable proliferation of MSCs, its stable cell phenotype and exist to osteogenesis, fat cells, such as multi-directional differentiation capacity, using virus transfection technology, for the first time will carry GFP report gene and Angl slow virus vector and efficient transfection human umbilical cord MSCs.2. Angl gene transfection in between umbilical cord mesenchymal stem cells can be reduced neutrophil lung infiltrates and alleviate edema and obviously improve LPS induced lung injury, relieve systemic inflammatory response caused by LPS;Improve the stem cells migrating to the inflammatory injury of lung tissue and the implant.3. LPS can lead to rat myocardial remodeling and left heart function obstacle, UC-MSCs-Angl after the intervention, inhibits inflammation injury of myocardial tissue factor and the expression of apoptosis related gene, reduce serum cTnl, content in different degree, restrain damage of myocardial tissue inflammatory reaction and apoptosis, significantly reduce the LPS induced rat LVEDP and dp/dt Max, increased LVEF and+dp/dt Max.Tips to improve left ventricular function. |
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