Relevant Research On Dushen Decoction Combined With Bone Marrow Mesenchymal Stem Cells In The Treatment Of Rats With Acute Myocardial Infarction

Part 1:Background: Stem cell is an emerging technology. It has self-renewal and multiplex differentiation potential in vitro. Stem cells could be used to treat various intractable diseases with cell transplanting method. Currently, BMSCs （Bone mesenchymal stem cells） attracts the most attention. But the content of BMSCs in bone marrow is in extremely low level, maybe only 2-5 BMSCs exit in every 100000 mononuclear cells. However, scientific investigations need enough, high purity BMSCs, so effective amplification in vitro become urgent need. Therefore, using effective separation, purification and amplification methods is particularly important.Objective: At present, commonly used methods for BMSCs separation, purification and amplification include the adhesive method, density gradient centrifugation, flow cytometric analysis, magnetic-activated cell sorting, special culture medium screening method and osmotic pressure screening method. And each method has its merits. According to the characteristics of various methods, combining research needs, our investigation aimed to explore and verify an effective separation, purification and amplification methods in vitro cells.Methods: BMSCs was cultured in vitro by density gradient centrifugation method combined with adhesive method.1. Take 1 Wistar rat every time to break its neck to death, infused it in 75% ethanol for 10 minutes. Eliminate femur and tibia in sterile conditions, removed the tissues adhere to bone surface, and rinse off with sterile PBS （phosphate balanced solution）. Remove two terminals of bones to expose the marrow cavity. Then removed joint surfaces and rinsed bone marrow cavity repeatedly with PBS by syringe （60mL）.2. Collected bone marrow and pour it into sterile centrifugal tube with 50ml DMEM （Dulbecco’s Modified Eagle Medium）, pumped repeatedly to make the tissues become cell suspension. Overlaid the cell suspension on lymphocyte separation liquid（1.077 Ficoll）to be centrifuged for 20 minutes （2000r/min）, and absorbed the single cell layer. Rinsed cells twice with DMEM for centrifugation for 5 minutes （1000r/min）, and discarded supernatant to take cells deposits. Added DMEM containing 15% volume fraction fetal calf serum into cells deposits to adjust the cell density to 1×10⁶/mL, and vaccinated it in 25cm² cell culture flask to culture in constant temperature cell incubator （37℃, saturated humidity CO₂ 5% volume fraction）. Replaced the medium after 48 hours, remove the cells not adhere to the cell flask. Afterwards, change 1 times every 3 days.3. If 80% of the original generation cells have been fusion, then passage it, and rinse off with PBS for 3 times. 0.25% Trypsin was added for digestion for 5 minutes then suspended digestion by adding it into DMEM medium containing 15% fetal calf serum when the forms of cells went round. Percussed it gently with straw, and put digest fluid into 15mL sterile centrifugal tube for centrifugation for 10 minutes （1000r/min）. Discarded supernatant and take cells deposits. Added them into DMEM containing 15%volume fraction fetal calf serum, passaged it by 1:2 proportion.Evaluation: then the surface markers of BMSCs, including CD44, CD90, CD34 and CD45, were determined by flow cytometry.Results:1. The morphologic observation of BMSCs showed that: under microscope, newly isolated vaccinal cells presented round, large mount, and different in size; 48 hours laer, most of the cells grew up adhering to the culture flask, and presented round or oval, increasing in size, and most of them were mononuclear cells, after this, spindle cells engendered; 7 days later, the cells presented fusiformis, and colony formation was obvious, and it mixed togethered to circinate or radial pattern as the unceasingly expands of colony formation; Primary cells fusion was up to over 85%, cells fusion after subculturing was up to over 85% after about 1 weeks; Cells passaged to 5^th generation presented consistent fusiformis.2.Flow cytometry of BMSCs showed that: CD44 positive cells take up 92.4%, CD90 positive cells take up 88.9%, CD34 positive cells account for 88.9%, and CD45 negative cells take up 95.6%, CD45 negative cells account for 95.6%.Conclusion: The experiment selected CD34, CD44, CD45 and CD90 to identify and testify the BMSCs cultured. Adherent screening method and density gradient centrifugation methods were used to obtain enough and quantity of BMSCs, which laid the foundation for the next step experiments in vivo. Part 2:Background: The improvement level of heart function after acute myocardial infarction directly related to its prognosis. How to better improve heart function after acute myocardial infarction attracts hot attention recently. It is found in a study that traditional Chinese medicine combining with the transplantation of BMSCs （Bone mesenchymal stem cells） could develop the synergistic and complementary function. However, it is under discussion that choosing which traditional Chinese medicine for intervention could play significantly improvement function.Objective: To observe the effect of Du-Shen decoction combined with BMSCs on the heart function of acute myocardial infarction in rats.Method: Choose 40 female Wistar rats in clean level, ligatured anterior descending branch of the left coronary artery to construct the acute myocardial infarction models with Olivette method. Rats models constructed were randomly divided into four groups: （1） acute myocardial infarction control group （n = 10） : rats were injected with equivalent medium after myocardial infarction; （2） BMSCs transplantation group （n = 10）; acute myocardial infarction rats accepted allogeneic treatment of BMSCs transplantation （2x10⁶ BMSCs/100u1, injected at 4 time points）; （3） Du-Shen decoction group （n = 10）: rats were forced to take Du-Shen decoction after acute myocardial infarction for 2 hours, twice a day for 1 week; （4） BMSCs transplantation + Du-Shen decoction group （n = 10）: acute myocardial infarction rats accepted allogeneic treatment of BMSCs transplantation （2x10⁶ BMSCs/100 u1, injected at 4 time points）, 2 hours later, the rats were forced to take Du-Shen decoction, twice a day for 1 week. At 4 weeks before modeling and after BMSCs transplantation, rats were examined with cardiac ultrasound, including left ventricular ejection fraction （LVEF） and cardiac minute output （CO）; after that, the cardiac muscular tissues of rats in all groups underwent HE staining. Results:1. Echocardiography results:1.1 LVEF results showed:①compared to pre-operation parameters, every post-operation parameter in AMI control group, Du-Shen decoction, BMSCs and BMSCs+Du-Shen decoction groups decreased obviously （P<0.05）;②Compared to the control group, every parameters in BMSCs, Du-Shen decoction and BMSCs+Du-Shen decoction groups increased;③There existed significant difference between BMSCs and AMI control groups （P<0.05）.④There existed significant difference between BMSCs+Du-Shen decoction and AMI control groups（P<0.01）, between BMSCs+Du-Shen decoction and BMSCs groups（P<0.05）, and between BMSCs+Du-Shen decoction and Du-Shen decoction groups（P<0.01）.1.2 CO results showed that:①compared to pre-operation parameters, every post-operation parameter in AMI control group, BMSCs, Du-Shen decoction and BMSCs+Du-Shen decoction groups decreased obviously with significant differences （P < 0.05）.②Compared to the control group, every parameter in BMSCs, Du-Shen decoction and BMSCs+Du-Shen decoction groups increased;③The parameters in BMSCs were much higher than that in AMI control group with significant difference （P < 0.05）.④The heart function of AMI rats in BMSCs+Du-Shen decoction group improved best. There existed significant difference between BMSCs+Du-Shen decoction and AMI control groups （P < 0.01）.2. Cardiac muscular tissues histopathology results:（1） In the control group, the rats’heart samples after HE staining showed extensive wide range of myocardia fibrosis, disorganized myocardia with wither or loose of adjacent myocardia fibrosis, a large number of scar tissues among cardiac muscular tissues, mild lymphocytes-dominated inflammatory cells infiltration and liparoid change.（2） In BMSCs groups, the rats’heart samples after HE staining showed that among cardiac muscular tissues, there existed connective tissue hyperplasia but no hemangioma found, and large number of small blood vessels. Conclusions:1. Du-Shen decoction could effectively promote BMSCs to improve heart function of AMI rats.2. Du-Shen decoction and BMSCs have good synergistic effect on improving heart function of AMI rats. Part 3:Background: Early experiments showed that Du-Shen decoction combined with BMSCs could improve heart function of AMI rats significantly. Currently, the following two aspects might be the reasons to improve heart function: firstly, Du-Shen decoction could improve the differentiation of BMSCs into cardiac cells; secondly, it could promote heart function by improving endothelial function, promoting regeneration of micrangium and developing the blood supply of infarct and ischemia areas. However, improving the heart function by increasing numbers of cardiac need a large number of cardiocytes, and the survival rates of stem cells in transplantation area is much lower, so improvement of heart function might give priority to improve endothelial function.Objective: To observe whether Du-Shen decoction combined with BMSCs could promote the improvement of vascular endothelial function of AMI rats, and whether Du-Shen decoction and BMSCs have synergistic effect on promoting the improvement of vascular endothelial function by detecting VEGF, ICAM-1 and VCAM-1 of rats.Methods: Before sampling heart of AMI rats, opened appendectomy to take the artery blood from the abdominal aorta, then centrifuged the artery blood and stored it in 20℃for serum VEGF, ICAM-1 and VCAM-1 detection （specific operation procedure strictly according to kit protocol）; And VEGF was detected by Western Blot.Results:1. Serum VEGF detection results were in line with VEGF Western results: （1） Compared to the control group, every parameters in BMSCs, Du-Shen decoction and BMSCs+Du-Shen decoction groups increased. （2） Compared to the control group, every parameter in BMSCs and BMSCs+Du-Shen decoction groups increased （P < 0.01）. （3） Compared to BMSCs group, that in BMSCs+Du-Shen decoction group increased dramatcally （P < 0.01）.2. Serum ICAM - 1 and VCAM - 1 testing results showed that: （1） Compared to the control group, every parameters in BMSCs, Du-Shen decoction and BMSCs+Du-Shen decoction groups decreased. （2） Compared to the control group, every parameter in BMSCs group decreased significantly （P < 0.05）. （3） Compared to the control group, every parameter in BMSCs+Du-Shen decoction group decreased obviously （P < 0.01）.Conclusion:1. Du-Shen decoction combined with BMSCs could improve vascular endothelial function of AMI rats obviously, maybe because the improvement of heart function after AMI has intimate connection with the improvement of vascular endothelial function.2. Du-Shen decoction and BMSCs have synergistic effect on improving vascular endothelial function of AMI rats.