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.

BACKGROUND: The therapeutic effects of acupuncture on weight loss have been verified. How does the central neural system act on weight loss with acupuncture?What relationships are there between the central regulation and peripheral action?OBJECTIVE: To study the effect of acupuncture on feeding center of hypothalamus in obese cases.DESIGN: Randomized and controlled study in which the experimental animals were taken as the subjects.SETTING: Clinical medical college of a university of traditional chinese medicine.MATERIALS: The experiment was performed in Key Laboratory of Acupuncture and Moxibustion in Nanjing University of Traditional Chinese Medicine from June 1998 to January 2000. Fifty SD male rats were employed and randomized into acupuncture group, obese group and normal group.METHODS: The experimental obese model was prepared by feeding SD rats with high-fat forage. Microelectrode record of nerve cell and cerebral stereotaxic technique were applied, by the treatment with acupuncture on experimental obese rats, the electric activity (Hz) of nerve cell was observed at unit time in hunger center of lateral hypothalamus area(LHA) and satiety center of ventral medium nucleus of hypothalamus(VMH).MAIN OUTCOME MEASURES: Mass changes at the same stage before and after acupuncture in rats of every group and discharge frequency in LHA and VMH in rats of every group.RESULTS: Acupuncture reduced remarkably the excitability in LHA, the values of which in acupuncture, obese and normal groups were(9.4 ± 3.7),(21.4 ± 6. 8), (9. 1 ± 5.2) Hz respectively( P < 0. 01 ); improved electric activity frequency in VMH, the values of which in 3 groups were (21.1 ± 4. 3), (5.0 ± 1.3), ( 14. 5 ± 2. 2)Hz, respectively( P < 0.01 ), inhibited excessive appetite of obese rats and reduced calorie intake so as to achieve weight loss.CONCLUSION: The regulation of acupuncture on central neural nuclei in obese animals is one of the important mechanisms on weight loss.

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 evaluate the influence of antiplatelet therapy prior to intravenous thrombolysis (IVT) on acute ischemic stroke (AIS) patients receiving IVT with recombinant tissue type plasminogen activator (rt-PA).Methods Researches about the safety of pre-existing antiplatelet treatment on AIS patients undergoing rt-PA IVT published before 31st December 2013 were retrieved based on internet databases.A meta-analysis of included clinical trials was performed by RevMan 5.2 and Stata 12.0 software.Simultaneously,funnel plot and Egger's test were used to evaluate the publication bias.Results A total of 10 papers were included.Eight researches based meta-analysis showed that pre-existing antiplatelet therapy increased the risk of symptomatic intracranial hemorrhage (SICH ; OR =1.67,95％ CI 1.44-1.93,P ＜ 0.01),6 researches based analysis suggested pre-existing antiplatelet therapy increased the risk of any intracranial hemorrhage (ICH ; OR =1.23,95％ CI 1.04-1.47,P ＜ 0.05) and 3 trials based analysis indicated the functional independence of patients receiving antiplatelet treatment was a bit worse than control group (OR =0.86,95％ CI0.80-0.93,P ＜0.01).Funnel plots and Egger' s test showed that there was no significant publication bias (P ＞ 0.05).Conclusions Antiplatelet therapy might increase the risk of post thrombolysis SICH and ICH,and their 3-month function independence is not so satisfied as those who had no antiplatelet agents before IVT.However,this review has limitations and the above results should be validated in future large prospective clinical studies.

Objective:To investigate if acupuncture has an inhibitory effect on fatty toxicity and its possible mechanism. Methods: Thirty-four patients with simple obesity were clinically treated with acupuncture for 3 courses. Before and after treatment, body mass index(BMI) and fat percentage(F%) were determined, and serum insulin and adiponectin were measured by enzyme linked immunoassay, blood lipid and sugar by biochemical colorimetry and eight kinds of free fatty acids(FFAs) by high performance liquid chromatography. Twenty normal persons were selected as a control. Results:The total efficacy rate was 88.2%. Acupuncture could increase the decreased insulin sensitivity index(ISI) in the patients(t=-5.103, P=0.000). The correlation of a decrease in F% with an increase in unsaturated fatty acid after acupuncture was of significance(r=0.402, P=0.019) and its correlation with an increase in the ratio between unsaturated fatty acid and saturated fatty acid was also of significance(r=0.348, P=0.044). The correlation between increases in high density lipoprotein and in eicosapentaenoic acid was of significance(r=0.352, P＝0.041). The correlation between a decrease in BMI and an increase in high density lipoprotein was of significance(r=0.357, P=0.038). Adiponectin level significantly rose after acupuncture (compared with pre-treatment, P=0.000). The correlation between a decrease in FFAs and an increase in adiponectin was of significance(r=-0.349, P=0.043). Conclusion: Acupuncture can lower the levels of free fatty acids in the patients and increase their sensitivity to insulin to inhibit the fatty toxicity. The inhibitory effect of acupuncture on fatty toxicity is somewhat related to a rise in adiponectin level.

Objective To explore the effects of multidisciplinary comprehensive pulmonary rehabilitation on aged patients with pneumo-coniosis. Methods From January, 2012 to January, 2015, 85 old stable inpatients with pneumoconiosis were divided into control group (n=40) and rehabilitation group (n=45). Both groups accepted routine medicine, while the rehabilitation group received multidisciplinary com-prehensive pulmonary rehabilitation for twelve weeks. They were assessed with cardiopulmonary exercise test, BODE index and quality of life before and after treatment. Results The maximum work, maximal oxygen uptake, 6-minute walk distance, scores of modified Version of the British Medical Researsh Council Respiratory Questionnaire, BODE index, activities of daily living, social activities, depression and anx-iety improved in the rehabilitation group after treatment (t>3.379, P<0.01), and were better than those in the control group (t>2.201, P<0.05). No imporvement was found in all the indices in the control group after treatment (t<2.339, P>0.05). Conclusion Multidisciplinary comprehensive pulmonary rehabilitation can effectively improve exercise tolerance and quality of life in aged patients with pneumoconiosis.

Objective: To investigate the proper methods of acupuncture and moxibustion for controlling body weight and their objects. Methods: 274 outpatients were allocated to 3observation groups: obesity, overweight and normal, using standard weight as an index. The obesity group was divided, at a ratio of 1: 1: 3, into 3 groups: fixed prescription, local treatment and treatment based on syndrome differentiation. The syndromes were classified into five types in the syndrome differentiation group. Different protocols of acupuncture and moxibustion plus auricular-plaster therapy were drawn up for treatment. Body mass index (BMI), body fat (F%),body circumferences and subcutaneous fat were evaluated in the patients before and after treatments. Results: There was a difference in the controlling effect of acupuncture on body weight among different populations. The curative effect was better in the obesity and overweight groups than in the normal group; the effective rate was 72.2%, 72.5% and 44.0%, respectively.There was also a difference in curative effect among obesity patients with different types of syndromes. The curative effects on retention of damp-heat in the stomach, stagnation of liver qi and dampness retention due to splenic hypofunction were equal but all better than those on deficiency of splenonephric yang and intemal heat due to yin deficiency. There was also a difference in curative effect among different protocols. The curative effects in the syndrome differentiation-based treatment and fixed prescription groups were equal (77.8%) but all better than that in the local treatment group (53.3%). Conclusion: Acupuncture and moxibustion is an effective way to treat obesity and overweight. Treatment based on syndrome differentiation and fixed prescription on the basis of syndrome differentiation are effective methods.

BACKGROUND: The abnormality of the function of arcuate nucleus may be an important factor of obesity. It has been known that the mechanisms of acupuncture in treating obesity are related to nervous and neurohumoral regulation. What is the regulating effect of acupuncture on the function of arcuate nucleus?OBJECTIVE: To study the effects of acupuncture on the function of arcuate nucleus of obese rats, and further investigate central nervous functional mechanism of reducing weight by acupuncture.DESIGN: Randomized controlled study based on the experimental animals.SETTING: Acupuncture institute in second clinical medical college of a university of traditional Chinese medicine, and a population management college.MATERIALS: This experiment was carried out in the Acupuncture Institute of Second Clinical Medical College, Nanjing University of Traditional Chinese Medicine between April and October 2002. One-month old male SD rats just in ablactation were selected.METHODS: Rats fed with ordinary wholesome rat-feed were in the normal group. The successfully established experimental obese rats models were randomly divided as control group and acupuncture group with 12 rats in each group. Rats in the acupuncture group were given acupuncture treatment for 14 days, and rats in the normal and control groups were put into rat fixation-machine for 15 minutes every day, lasting for 14 days. Body mass, Lee' s index, body lipid, level of central and peripheral leptin and insulin(INS) as well as the frequency of spontaneous discharge of nerve cell in the arcuate nucleus(ARC) of hypothalamus in obese rats were observed with nervous electrophysiological and nervous biochemical technology before and after acupuncture.MAIN OUTCOME MEASURES: ① Effect of acupuncture on obesity index, fat contents of pericardium, kidney and epididymis of experimental obese rats. ② Effect of acupuncture on the frequency of spontaneous discharge of ARC nerve cell of hypothalamus in obese rats. ③ Effect of acupuncture on the contents of hypothalamic and serum leptin and INS of rats in each groups. ④ Correlation of the frequency of spontaneous discharge of ARC nerve cell of hypothalamus with the level of hypothalamic leptin and INS.RESULTS: The body mass[(517.74±29.35) g], Lee' s index (319.85 ± 3.96) and body fat content of obesity rats [ (13.88 ± 1.32) g] were all significantly higher than those in the normal rats, the differences were significant(P ＜ 0.01); the frequency of spontaneous discharge of nerve cell in ARC of hypothalamus in obese rats [ (3.12 ± 1.92) Hz] were all markedly lower than those of the normal rats[ (8.99 ± 2.71 ) Hz], the differences were significant (P ＜ 0.01 ); the frequency of spontaneous discharge of nerve cell in ARC was negatively correlated with body mass, Lee' s index and body lipid( r = -0. 592, -0. 672, -0. 521 ) . Acupuncture significantly increased the frequency of spontaneous discharge of nerve cell in ARC of hypothalamus in obese rats [(9.75 ±2.02) Hz] as compared with the control group, the differences were significant( P ＜ 0. 01 ).CONCLUSION: The good regulating effect of acupuncture on the function of arcuate nucleus of hypothalamus might be one of mechanisms in the antiobesity of acupuncture.

Objective: To select suitable conditions for prokaryotic expression and purification of rhIL-17. Methods: rhIL-17 was expressed in E. coli host under heat induction. After compared among the expression amounts in different media under different heat induction time, the most suitable conditions was selected. The target protein was present in the form of inclusion body. The precipitate of inclusion was obtained and purified after 6M guanidine solublization or 2% SDS solublization. Results: Either protocol could yield rhIL-17 with high purity and stable activity. The SDS solublization mehthod gives rise to much more higher productivity than the guanidine solublization method. Conclusion: rhIL-17 were expression in E. coli system and purified to homogenicity by SDS solublization methods with high productivity.

Aim To construct and express anti-CD20Fab-LDP,generate anti-CD20Fab-LDM and identify its biological activity.Methods PCR and overlapping PCR were used to construct anti-CD20Fab-LDP.DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS.Specific killing activity in vitro of anti-CD20Fab-LDM was analyzed by MTT.Results The data of DNA sequence showed that anti-CD20Fab-LDP was correct.The fusion protein was recovered in high yield(up to 4 mg·L~(-1))after proteinG purification.The fusion protein could bind to Raji cells(CD20+),and similar affinity data were obtained with anti-CD20Fab.Anti-CD20Fab-LDP showed potent cytotoxicity to Raji cells with IC_(50) values of 0.9×10~(-10) mol·L~(-1).Conclusions Anti-CD20Fab-LDP with high level expression was successfully obtained and could bind to Raji cells cells.Anti-CD20Fab-LDM showed specific killing activity to Raji cells in vitro.