BACKGROUND:Mesenchymal stem cel s as potential seeded cel s have been widely used in tissue engineering and clinic therapy;thus, the precise, safe, effective isolation of mesenchymal stem cel s is the particular important premise to build culture system.
OBJECTIVE:To review the methods of isolating mesenchymal stem cel s and to compare the merit and demerit of different methods, thereby providing theoretical basis for safe and high-effective isolation of mesenchymal stem cel s.
METHODS:A computer-based online research of CNKI and PubMed databases was performed to col ect articles, which included reviews, clinical trials and experiments, published between 1965 and 2014 with the key words of“mesenchymal stem cel s (MSCs), isolation methods”in Chinese and English. A total of 52 articles were included according inclusion and exclusion criteria
RESULTS AND CONCLUSION:(1) The whole bone marrow culture method can derive a mass of mesenchymal stem cel s, which need to be purified. (2) The density gradient centrifugation method which uses the media with the density of 1.073 g/mL can be used to harvest more purified cel s. (3) The tissue digestion method is suitable for digestion and isolation of adipose tissue and umbilical cord tissue. Type II col agenase digestion is better, but they are both limited by a high demand for operative techniques. (4) Immunomagnetic bead separation is appropriate to study the biological characteristics of a kind of subpopulation of mesenchymal stem cel s which express special surface markers. (5) The combination method is also an optimal way. (6) Some new methods limited by few dates require further studies.

Objective To investigate the methods and efficacy of coronary stent placement in patients with myocardial bridge. Methods 4 patients with angina and ST-T changes in ECG were involved in the study. Coronary angiography showed there were 75%～90% systolic stenosis of LAD in all patients. Two of them were regarded as isolated myocardial bridge, and the others were regarded as myocardial bridge associated with atherosclerosis. The length of artery pressed by myocardial bridge was in the range of 10～18 mm. All patients who had repeated angina accepted stenting therapy. One of the stents was Crossflex, three were Nir stents. Stents size: (3.0～3.5)?(12～20)mm. All stents were expanded with 8～10 atm for 30～40 seconds. Results After stenting, coronary angiography showed no residual stenosis. In the follow-up 6～18 months there were no complaints of angina. In two of them, coronary angiography showed there were no restenosis after one year.Conclusion Coronary stenting is an optional method to treat patient who have myocardial bridge and have no efficacy after taking medicines, especially for the patients who have both myocardial bridge and atherosclersis. But futher study in large populations is still necessary.

BACKGROUND:In previous studies, it is satisfied to sorting bone marrow mesenchymal stem cels based on natural sedimentation combined with low permeation. Then, based on particle hydromechanics theory, the settling velocity of bone marrow mesenchymal stem cels in culture liquid is calculated. A simple and easy method of separation, purification and proliferation of bone marrow mesenchymal stem cels is established by natural sedimentation velocity. OBJECTIVE:To explore the feasibility of sorting bone marrow mesenchymal stem cels by natural sedimentation velocity method. METHODS:The density interval of rabbit bone marrow mesenchymal stemcels (ρ1) was determined by density gradient centrifugation method. The diameter of rabbit bone marrow mesenchymal stem cels (d) was measured by scanning electron microscope. The density of culture liquid (ρ2) was measured by liquid density meter. The viscosity of the culture liquid (μ) was measured by viscosity meter. The settling velocity of bone marrow mesenchymal stem cels (Vt) was derived from the above four numerical values with the appropriate formula. Bone marrow mesenchymal stem cels were sorted from bone marrow tissue by natural sedimentation velocity method. Cel proliferation, purity and differentiation were observed. Meanwhile, the primary culture time of three different cel sorting methods was recorded; the colony formation rate of rabbit bone marrow mesenchymal stem cels was determined. RESULTS AND CONCLUSION:(1) The diameter of rabbit bone marrow mesenchymal stem cels was (20.37±4.58) μm, and the setting velocity of rabbit bone marrow mesenchymal stem cels in the culture liquid was 50-55 mm/h. (2) Bone marrow mesenchymal stem cels could be successfuly isolated from bone marrow tissue of rabbits by the natural sedimentation velocity method, which could be induced into osteoblasts and adipocytes. (3) The natural sedimentation velocity group cost less time than the other two density gradient centrifugation groups in the primary culture stage. The colony formation rate of the natural sedimentation velocity group was higher than that of the other two groups. (4) Natural sedimentation velocity method did not impose any intervention measures for sorting cels, which maybe maximaly maintain cel viability and biological characteristics. The whole separation process was simple and safe, which may have little damage to the cels.

Objective To investigate the effects of cognitive impairment in hippocampus on glucose and lipid metabolism, and its relations with gastrointestinal motility. Methods The Aβ1-42 was injected into the hippocampus of rats. Levels of glucose and lipid were detected. The changes of gastrointestinal motility were detected by the type-B ultrasonic and the ink-pushing experiments. Hippocampal neurons apoptosis was detected by the TUNEL assay. Results In the experimental group, FPG, TG, TC, LDL were (7.92 ± 0.29) mmol/L, (2.24 ± 0.12) mmol/L, (4.67 ± 0.12) mmol/L, (2.41 ± 0.12) mmol/L, respectively, with significant differences among these three groups (P < 0.05). Compared with the sham group and the control group, the number of bowel movements per unit time (2.13 ± 0.83) times, gastric emptying rate (44.35 ± 7.53) % and the small intestinal propulsion rate (57.60 ± 7.82)%in the experimental group were significantly decreased (P<0.05). The experimental hippocampal neuronal apoptosis index was an average of (64.98 ± 3.70)%, which was significantly higher than that in the sham group and the control group (P < 0.05). Conclusion Hippocampal cognitive impairment can elevate the blood lipid level, which may be associated with the hippocampal neuronal apoptosis and the gastrointestinal motility disorders.

BACKGROUND:Thoracic vertebra connected to the corresponding section of the ribs constitute thoracic vertebral, which is deep, and the structure is complicated, so it is difficult to ful y expose thoracic vertebrae. Usual y, corresponding ribs is removed, and the injured site wil be reached through thoracic cavity. The trauma is big. Some complications often occur such as chest pain, and local skin numbness. Therefore, whether it is possible to reach the same target without removal of ribs through intercostal space became a new clinical problem. OBJECTIVE:To explore the safety and efficacy of autologous iliac bone graft and plate fixation for tuberculosis of thoracic vertebra. METHODS:A total of 30 patients diagnosed with tuberculosis of thoracic vertebra from January 2008 to December 2013 were conventional y treated with anti-tuberculosis treatment for 2 to 3 weeks, and then treated with autologous iliac bone fusion through intercostal space and anterior plate fixation. Postoperative fol ow-up was conducted from 6 to 22 months. Fracture healing condition, the degree of pain relief, Cobb angle change, length of incision, blood loss, operation time, postoperative recovery of neurological function were observed. RESULTS AND CONCLUSION:In 30 patients, the length of incision was (12.4±1.8) cm;longitudinal incision distraction width was (10±3.2) cm;the time of opening the chest was (16.0±2 .5) minutes;the time of closing the chest was (12.0±1.5) minutes;intraoperative blood loss amount was (430.0±87.4) mL. Preoperative and postoperative average kyphosis angles were respectively 27° and 8°, with an average rectification of 19°. The pain basical y relieved at 1 to 2 weeks after the surgery. 28 patients were healed, and the symptoms of 2 patients were improved. Postoperative fol ow-up radiographs revealed that autologous bone grafts were thoroughly fused, and the fusion time lasted from 4 to 5 months. These data verified that autologous iliac bone graft through intercostal space and plate fixation is an effective, safe method for tuberculosis of thoracic vertebra. The exposed range through intercostal space can satisfy the operation requirements of complete tuberculosis clearance, autologous iliac bone graft and plate fixation, and can ensure the integrity of the whole thorax and spine stability.

AIM: To investigate alteration of inward re ctifier potassium current (I K1) in atrial myocytes and mRNA expression of gene Kir2.1 encoding I K1 in atrial myocardial tissue in patients with chronic atrial fibrillation (AF) compared to that with sinus rhythm (SR).METHODS: Single myocytes were isolated by enzymatic dissociation with the chunk method an d the ionic current was recorded using whole cell patch clamp technique. The sem i-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of Kir2.1 in atrial myocardial tissue, and the g ene GAPDH was used as an internal control.RESULTS: (1) The I K1 density was increased in AF group at hyperpolarizing pot entials, at -120 mV the current densities was (-5.71?0.65) pA/pF in AF group (n=28 cells from 7 patients) and (-4.26?1.22) pA/pF in SR group (n=35 cells from 9 patients) (P0.05).CONCLUSIONS: The increase in I K1 at hyperpolarizing potentials may be related to th e atrial electrophysiological remodeling in chronic human AF. The increased I K1 density in atrial myocytes in AF group without alteration of Kir2.1 mRNA expression in atrial tissue suggests that I K1 may be mediated at post-transcriptional levels.

Objective:To explore the alteration of brain‐derived neurotrophic factor (BDNF) signaling and the influ‐ence of irbesartan on it in hippocampus of angiotensin‐converting enzyme 2 (ACE2) knock‐out (KO) mice . Meth‐ods:The 10~11‐week ACE2 KO (Ace2/y ) mice received daily treatment with angiotensin II (Ang II) type 1 (AT1) receptor blocker irbesartan (50 mg/kg) or placebo for two weeks. The wild‐type mice (WT ,Ace2+ /y ) were regarded as normal control. Western blotting method was used to measure levels of BDNF and extracellular signal regulated kinase 1/2 (ERK1/2) in the mice hippocampus. Radioimmunoassay was used to measure plasma Ang level in mice . Results :Compared with normal WT control mice ,there were significant down‐regulations of BDNF protein expres‐sion [ (1 ± 0.16) vs .(0.54 ± 0.16)] in hippocampus and plasma Ang‐ (1‐7) level [ (55.6 ± 7.5) pg/ml vs .(42.8 ± 5.8) pg/ml] ,and significant rise in ERK1/2 phosphorylation [ (1 ± 0.28) vs .(1.79 ± 0.29)] in ACE2 KO mice (P<0.01 all). After irbesartan treatment ,there were significant rise in BDNF protein expression (0.88 ± 0.13) in hippocampus and plasma Ang‐ (1‐7) level [(59.4 ± 8.4) pg/ml] ,and significant reduction in ERK1/2 phosphoryla‐tion level (1.33 ± 0.19) in ACE2 KO mice (P<0.05 or <0.01) .Conclusion:There are BDNF protein expression down‐regulation and enhanced ERK1/2 phosphorylation in hippocampus of ACE2 KO mice. AT1 receptor blockade irbesartan can improve Ang‐ (1‐7 ) level and hippocampus BDNF expression , while reducing hippocampus ERK phosphorylation signal in ACE2 KO mice ,suggesting that AT1 receptor blockade possesses certain brain protective effect.

ObjectiveTo study the physiologic and pathlogic alteration of the artificial bile duct generated by pedicled jejunum flap(CBD)in repairing local defect common bile duct of dog. Methods Common bile duct defected model was generated by partially cutting off CBDofdog.The pedicled jejunum flap with mesentery blood supply was created to repair the local defect CBD.The pH of bile was mcasuratcd during and post operation, the pathologic study was undertaken after 6 months.ResultsThe experimental group live 6 months and the symptom and signs is abnormal after operation.The result of blood routine and biochemistry examination is no change(P＞0.05).The pH of postoperative bile decrease(P＜0.01).The pH of bile was changed remarkably 1 day after the pedicled jejunum flap replacement.But no significant changing in the following 6 months(P＞0.05).The postoperative pathologic study show:the anastomosis of experimental group healing well,and fiber tissue proliferates at the site of anastomosis,The Epithelium of the jejunum flap was atrophy and there were large quantity of lymphocytes,plasma cells and neutrophil within the jejunum flap(10/10).The epithelium of bile duct nearby the jejunum flap appears slight proliferate and infused with lymphocytes and plasma cells(10/10).There were lymphoid follicles in 3/10 cases.ConclusionsThe jejunum flap repairing bile duct healing well,but there was inflamatic change around the jejunum flap and nearby bile duct after the replacement.These observation suggested it is feasible to use the jejunum flap repairing the defected bile duct.The local physiology is stable without any changes in pH of bile acid.The alterative materials like umbilical ligment,blood vessel would be favorable in repair of defected bile duct.

Objective: To evaluate the short-term effect of arthroscopic Endobutton combined with Orthocord suture fixation for tibia eminence fracture of anterior cruciate ligament in adults. Methods: The data of 9 adult patients with tibia eminence fracture of anterior cruciate ligament treated with Endobutton combined with Orthocord suture fixation from April 2017 to October 2018 were retrospectively analyzed. The range of motion of knee joint and the stability of knee joint were recorded 3 and 6 months after operation. Results: All patients were followed up for 3 and 6 months after operation. CT examination showed fracture reduction and healing 2 months after operation, and magnetic resonance image examination showed good anterior cruciate ligament tension 3 months after operation. The range of motion of knee joint was 0°-130° after 3 and 6 months, and Lanchman test was negative. The Lysholm score was (45.2 ± 6.7) scores, and IKDC score was (55.1 ± 7.3) scores before operation. The Lysholm score was (89.1 ± 7.2) scores, IKDC score was(87.4 ± 6.1) scores, Lysholm score was (95.4 ± 6.5) scores and IKDC score was (93.2 ± 7.8) scores at 6 months after operation. Conclusions Endobutton combined with Orthocord suture fixation under arthroscopy has a good short-term effect in the treatment tibia eminence fracture of anterior cruciate ligament in adults.

Objective To investigate the changes in the expression levels of miR-21 and its regulatory proteins in bone tissues of osteoporotic mouse model, and its correlation with bone mineral density. Methods Thirty ovariectomized (ovx) female mice were randomly divided into six groups according to different observation time (0 week, 1 week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks, respectively). Smad7 expression level in the tissues of the distal femur of these mice was immunohistochemically analyzed. Real time PCR was performed at different time points for detecting changes in the mRNA levels of miR-21 and Smad7 target genes in the bone tissue. Dual-energy X-ray absorptiometry (DXA) was used to measure the distal femoral bone density in mice of each group. Results Immunohistochemical study revealed that the expression of Smad7 in the bone tissue of osteoporostic mouse gradually increased over time. Real time PCR analysis showed that the expression of mi R-21 gradually decreased, whereas the expression of Smad7 gradually increased. Over time, the bone density of mice gradually decreased, indicating that miR-21 was positively correlated with bone density, whereas Smad7 was negatively correlated with bone density. Conclusion Bone density of the osteoporotic mice gradually decreases. The expression of miR-21 is positively correlated with bone density, while that of Smad7 is negatively correlated with bone density.