[Treating knee osteoarthritis by Chinese medicine and its correlation study with CT changes of infrapatellar fat pad].

OBJECTIVE: To observe the efficacy of Jianbu Tongluo Xunzheng Liquid (JTXL) in treating knee osteoarthritis (KOA), and to explore the correlation between changes of infrapatellar fat pad scanned by CT and the efficacy. METHODS: Totally 105 KOA outpatients were randomly assigned to three groups, i.e., the treatment group, the control group, and the combination group, 35 in each group. Patients in the treatment group were fumigated by JTXL, 30 min each time, once daily, 10 times as a course of treatment, 3 courses in total. Those in the control group received intra-articular injection of Sodium Hyaluronate Injection (SHI), 3 mL each time, once per 6 days, 5 times in total. Those in the combination group were treated by fumigation of JTXL + intra-articular injection of SHI in the same way as the aforesaid two groups. All patients were treated for 30 days. Their clinical efficacy and changes of infrapatellar fat pad scanned by CT were observed, and their correlation was analyzed. RESULTS: The total effective rate was 88.57% in the combination group, better than that of the control group (74.29%) and the treatment group (80.00%; both P < 0.05). Besides, the score for knee joint functions at Hospital for Special Surgery (HSS) was better in the combination group than the other two groups (P < 0.05). The anteroposterior diameter, exterior-interior diameter, the superior-inferior diameter were shortened, and the density decreased in the treatment group and the combination group (P < 0.05). Besides, they were superior to those of the control group (P < 0.05). CONCLUSIONS: Changes of infrapatellar fat pad scanned by CT only existed in the combination group and the treatment group, indicating changes of CT scanning was only correlated with effect on changing physicochemical properties of infrapatellar fat pad. Treatment by Chinese medicine could omnipotently and balanced regulate functions and structures of every tissue. Therefore, CT could be taken as a better method for clinical efficacy observation by Chinese medicine.

[Expressions of myogenic markers in skeletal muscle differentiation of human bone marrow mesenchymal stem cells].

OBJECTIVE: To investigate the expressions of myogenic markers MyoD, myogenin,and desmin in skeletal muscle differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). METHODS: Myogenic markers MyoD, myogenin,and desmin of hBM-MSCs cultured in vitro were detected by immunofluorescence and RT-PCR. A total of 21 8-to-10 week-old immunosuppressed mdx mice were transplanted with 1x107 passage 5 of hBM-MSCs. The mice were euthanized 2-24 weeks after transplantation,and gastrocnemius muscle were analyzed for human MyoD, myogenin,desmin,and dystrophin (Dys) expressions by immunohistochemistry and RT-PCR. RESULTS: The numbers of MyoD-,myogenin-,and desmin-positive cells per 100 hBM-MSCs were 23.5∓5.3, 30.7∓6.2, and 28.4∓5.7, respectively. MyoD, myogenin, and desmin mRNA was observed in passage 5 of hBM-MSCs. After two weeks of hBM-MSCs transplantation,a small number of MyoD-and myogenin-positive cells were observed in skeletal muscle of mdx mice,and desmin-positive cells were observed 4 weeks after transplantation. Expressions of MyoD and myogenin were detected in the muscle of mdx mice 2-4 weeks after hBM-MSCs transplantation, which reached a peak 12-16 weeks later. Desmin was expressed in the muscle of mdx mice 4-8 weeks after transplantation,with much more expression after 16 weeks of transplantation. A small number of Dys-positive cell and Dys mRNA expression were presented in the muscle of mdx mice 4 and 8 weeks after hBM-MSCs transplantation,respectively. The expression of Dys in the muscle of mdx mice increased gradually after transplantation. CONCLUSION: hBM-MSCs have the potential of myogenic differentiation in vitro and contribute to myogenic conversion in xenogeneic animal,during which the up-regulation of MyoD and myogenin expressions may play an important role.

[Mesenchymal stem cells transplanted in mdx mice differentiate into myocytes and express dystrophin/utrophin].

OBJECTIVE: To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into myocytes and their expression of dystrophin/utrophin after transplantation in mdx mice. METHODS: BrdU-labeled fifth-passage rat MSCs were transplanted in mdx mice with previous total body gamma irradiation (7 Gy). At 4, 8, 12 and 16 weeks after the transplantation, the mice were sacrificed to detect dystrophin/BrdU and utrophin expressions in the gastrocnemius muscle using immunofluorescence assay, RT-PCR and Western blotting. Five normal C57 BL/6 mice and 5 mdx mice served as the positive and negative controls, respectively. RESULTS: Four weeks after MSC transplantation, less than 1% of the muscle fibers of the mdx mice expressed dystrophin, which increased to 15% at 16 weeks. Donor-derived nuclei were detected in both single and clusters of dystrophin-positive fibers. Some BrdU-positive nuclei were centrally located, and some peripherally within myofibers. Utrophin expression decreased over time after transplantation. CONCLUSION: The myofibers of mdx mice with MSC transplantation express dystrophin, which is derived partially from the transplanted MSCs. Dystrophin expression from the transplanted MSCs partially inhibits the upregulation of utrophin in mdx mouse muscle, showing a complementary relation between them.

[Dynamic distribution of implanted human bone marrow mesenchymal stem cells in mdx mice].

OBJECTIVE: To investigate the dynamic distribution of human bone marrow mesenchymal stem cells (hBM-MSCs) in mdx mice. METHODS: Twenty-four 8-10-week-old immunocompromised mdx mice were transplanted with 1 x 10(7) passage 5 hBM-MSCs labeled with bromodeoxyuridine (BrdU) by means of injection into the tail vein. The mice were euthanized 48 hours and 2, 4, 8, 12, 16, 20, and 24 weeks after transplantation. BrdU-positive cells in tissue and organs of the mice were detected by immunofluorescence analysis. Skeletal muscle was stained for anti-human nuclei mouse monoclonal antibody (anti-Hu) and analyzed for human dystrophin (Dys) expression by immunohistochemistry and reverse transcription-polymerase chain reaction. RESULTS: After transplantation, BrdU-positive cells were found in most organs (especially in bone marrow, liver, and lung) within 4 weeks, and these cells in liver and lung decreased gradually after 4 weeks. At 48 hours after transplantation, BrdU-positive cells were found in bone marrow, which reached a peak level after 2 weeks and were still detectable after 16 weeks. BrdU-positive cells in skeletal muscle increased gradually over time of transplantation. A small number of anti-Hu positive cells were detected in skeletal muscle 2 weeks after transplantation. A small number of Dys positive cell were seldom found at 4 weeks and small Dys mRNA expression detected 4 weeks after transplantation. The proportion of anti-Hu in parallel with Dys positive cells and Dys mRNA in skeletal muscle of mdx mice increased gradually over time of transplantation. CONCLUSION: After being transplanted into mdx mice, hBM-MSCs are mainly distributed in bone marrow, liver, and lung during the early time (2-4 weeks) , and then in bone marrow and skeletal muscle (after 4 weeks).

[Dystrophin expression in mdx mice after bone marrow stem cells transplantation].

OBJECTIVE: To investigate the dynamic changes of dystrophin expression in mdx mice after bone marrow stem cells transplantation. METHODS: The bone marrow stem cells of C57 BL/6 mice (aged 6 to 8 weeks) were injected intravenously into the mdx mice (aged 7 to 9 weeks), which were preconditioned with 7Gy gamma ray. The amount of dystrophin;expression in gastrocnemius was detected by immunofluorescence, reverse transcription-polymerase chain reaction and Western blot at week 5, 8, 12 and 16 after transplantation. RESULTS: At week 5 after bone marrow stem cells transplantation, the dystrophin expression detected in mdx mice were very low; however, its expression increased along with time. At week 16 week, about 12% muscle cells of all transplanted mice expressed dystrophin. There were less centrally placed myonuclei than the control mdx mice, whereas peripheral myonuclei increased. CONCLUSIONS: After having been injected into mdx mice, the allogenic bone marrow stem cells have a trend to reach the injured muscle tissues and differentiate to fibers that can express dystrophin and the expression increased with time. The bone marrow stem cells participates in the repair and regeneration of the injured tissues permanently and constantly.

[Dystrophin expression and pathology of diaphragm muscles of mdx mice after xenogenic bone marrow stem cell transplantation].

OBJECTIVE: To investigate the effect of bone marrow stem cell transplantation (BMT) on the diaphragm muscles of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD). METHODS: The bone marrow-derived stem cells form male SD rats was transplanted through the tail vein into 18 female 8-week-old mdx mice, which were sacrificed at 4, 8 and 12 weeks after BMT (6 at each time point), respectively. The diaphragm muscles of the mice were subjected to HE staining, immunofluorescence detection of dystrophin, reverse transcription (RT)-PCR analysis of dystrophin mRNA transcripts and PCR analysis of Sry (sex-determining region on the Y chromosome) gene, with age-matched female C57 mice and untreated mdx mice as the controls. RESULTS: The proportion of centrally nucleated fibers (CNF) in the diaphragm muscle of the recipient mdx mice was (15.58+/-0.91) %, (12.50+/-1.87) % and (10.17+/-1.17) % at 4, 8 and 12 weeks after BMT, respectively, significantly smaller than that of untreated mdx mice [(19.5+/-1.87) %], and the fibers after BMT showed less inflammatory infiltration. Compared with the untreated mice, the recipient mdx mice showed green fluorescence on significantly more diaphragm muscle cell membranes [with the proportion of dystrophin-positive fibers of (1.00+/-0.32) %, (6.00+/-1.05) % and (11.92+/-1.11) % at 4, 8, and 12 weeks after BMT]. RT-PCR of dystrophin mRNA also demonstrated significantly higher relative levels of dystrophin in the recipient mdx mice (0.19+/-0.05, 0.26+/-0.06 and 0.36+/-0.04 at 4, 8 and 12 weeks after BMT) than in untreated mdx mice, and Sry gene was present in the recipient mice. CONCLUSION: BMT can partially restore dystrophin expression and ameliorate the pathology in the diaphragm muscles of mdx mice, and has great potential to produce general therapeutic effect in patients with DMD.

[Transplantation of 3H-thymidine-labeled human bone marrow-derived mesenchymal stem cells in mdx mice].

OBJECTIVE: To investigate the feasibility of using human bone marrow-derived mesenchymal stem cells (hBM- MSCs) for repairing the skeletal muscle sarcolemma lesions in mdx mice and characterize the distribution of the transplanted hBM-MSCs. METHODS: Eighteen 8- to 10-week-old immunosuppressed mdx mice received transplantation with 1x10(7) of hBM-MSCs (the fifth passage) with 3H-thymidine (3H-TdR) labeling by injection of the cells into the tail vein. The mice were killed at 24 h, 48 h, 2 weeks, and 1, 2 and 4 months after the transplantation, respectively, to measure the radioactivity in the tissues and organs. Dystrophin expression on the sarcolemma was detected by immunofluorescence analysis. RESULTS: One month after transplantation, the mice with cell transplantation showed greater radioactivity in most of the tissues and organs than the control mice, especially in the bone marrow, liver and spleen. The radioactivity was then gradually lowered but in the skeletal muscle, the radioactivity increased progressively since 2 weeks after transplantation, reaching the peak of 27.65+/-3.53 Bq/mg at 1 month. Compared with that in the control mice, the radioactivity in the bone marrow and skeletal muscle was persistently higher in mice with cell transplantation 1 month after transplantation. No dystrophin-positive cells were found in the mdx mice at 2 weeks but detected at 1 month. The percentage of dystrophin-positive fibers in each section ranged from a 6.6% (1 month) to 8.9% (4 months). CONCLUSIONS: hBM-MSCs engrafted in immunosuppressed mdx mice may differentiate into skeletal muscle cells to repair the pathological lesion of the skeletal muscle sarcolemma. The hBM-MSCs reside mainly in the bone marrow, liver and spleen in the early stage following transplantation, homing into the bone marrow and skeletal muscle later.

[Induced differentiation of human cord blood monocytes into neuron-like cells in vitro].

OBJECTIVE: To probe the conditions for inducing human cord blood monocytes to differentiate into neuron-like cells. METHODS: The mononuclear cells were isolated from human umbilical cord blood samples and plated in 25-mm culture flasks containing DMEM/F12 medium. The fifth passage of mesenchymal stem cells (MSCs) were induced by beta-mercaptoe- thanol (beta-ME), dimethyl sulfoxide (DMSO) and conditioned medium for neuron induction, respectively, to differentiate into neuron-like cells. The expression of nestin, neuron-specific enolase (NSE) and neurofilament (NF) on the treated cells were detected by immunocytochemical method. RESULTS: Nestin expression were found in 6.7% of the primary, 12.4% of the second passage, 20.8% of the fifth passage of MSCs, respectively. Thirty-six percent of cell cultures treated with the conditioned medium for neuron induction were immunoreactive for NSE and NF, and 33% of the cells induced by beta-ME and 25% of the cells by DMSO also expressed NSE and NF. CONCLUSION: Cord blood MSCs possess some features of neural stem cells, and have the capacity to differentiate into neuron-like cells under proper conditions.

[In vitro bromodeoxyuridine labeling of rat bone marrow-derived mesenchymal stem cells].

OBJECTIVE: To study the optimal dosage and timing for bromodeoxyuridine (BrdU) labeling of rat bone marrow-derived mesenchymal stem cells (MSCs) in vitro. METHODS: Bone marrow-derived MSCs of SD rats were cultured in vitro routinely and the sixth passage was taken for identification of specific surface antigens by flow cytometry. Before reaching cell confluence, the purified MSCs were incubated with BrdU at different concentrations (5, 10, and 15 micromol/L) for different incubating time (3, 12, 24, 36, 48, and 72 h) with 10 micromol/L BrdU, to identify the optimal BrdU concentration and incubating time for cell labeling. Immunohistochemistry was performed to calculate the labeling index (LI). RESULT: Flow cytometry showed that MSCs expressed CD29 and CD44 but not CD11b or CD45. Incubation of the MSCs with BrdU at 10 micromol/L and for an optimal length of 48 h appeared to achieve the highest LI, both of which exceeded 98%, with the labeling identifiable in five consecutive passages. CONCLUSIONS: The continually passaged cells are MSCs, the incubation of which with 10 micromol/L BrdU for 48 h may achieve a LI over 98% without producing obvious cell damages. The results suggest that BrdU labeling provides a feasible means for a dynamic in vivo observation of the survival, growth and differentiation of the implanted MSCs.

[Brain-derived neurotrophic factor induces rat bone marrow stromal cells to differentiate into neuron-like cells in vitro].

OBJECTIVE: To investigate brain-derived neurotrophic factor (BDNF)-induced differentiation of rat bone marrow stromal cells (MSCs) into neuron-like cells in vitro and observe its neuroprotective effect of BNDF on the differentiated cells, which might provide the better seed cells for treatment of nervous system diseases. METHODS: The fifth-passage MSCs were induced by BDNF and 2-mercapto ethanol beta-ME respectively, 1, 3 and 6 h after which the induced neuron-like cells were counted and compared. At 3 h, the neuron-like cells were identified by the immunocytochemical staining, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: The two induced cells both displayed neuronal morphologies with long and multipolar cell projections, but BDNF-induced cells survived for a longer time than beta-ME-induced ones. The results of immunocytochemical staining showed that the two neuron-like cells expressed nestin, neuron-specific enolase (NSE), neurofilament (NF), microtubule-associated protein (MAP-2), but not glial fibrillary acidic protein (GFAP). RT-PCR detected mRNA-positive NSE, NF and MAP-2 in the induced cells, with also mild positive GFAP mRNA. Western blotting identified also NSE expression in these neuron-like cells. CONCLUSION: BDNF alone may induce rat MSCs to differentiate into neuron-like cells in vitro, which have longer lifetime to better serve the purpose of transplantation and gene therapy for nervous system diseases.

[Dynamic changes in the expressions of myogenic regulatory factors MyoD and myogenin during repair of muscle injury].

OBJECTIVE: To observe the dynamic changes in the expressions of myogenic regulatory factors MyoD and myogenin during the repair of injured muscle. METHODS: Muscular injury model was established by local injection of bupivacain, and at different time points following the injection, the gastrocnemius muscles were collected for preparation of cryosections. HE staining was performed for examination of the pathological changes in the injured muscles, and the expressions of MyoD and myogenin were detected by SABC. RESULTS: MyoD-positive nuclei began to appear 18 h after muscle injury, reaching the peak till 48 h after the injury. Myogenin-positive nuclei appeared 24 h after muscle injury and peaked at 72 h. CONCLUSIONS: MyoD and myogenin play a role in muscle regeneration after muscle injury, and they may serve as the indexes to identify muscle precursor cells and mark muscle regeneration process.