OBJECTIVE To observe the clinical efficacy and safety of tofacitinib combined with hydroxychloroquine in the treatment of refractory rheumatoid arthritis （RA）. METHODS From January 1， 2021 to January 1， 2022， 120 patients with refractory RA were selected as the study objects. According to the principle of random allocation， the patients were divided into group A， group B and group C， with 40 patients in each group. Group A was given Tofacitinib citrate tablet + Hydroxychloroquine sulfate tablet； group B was given Tofacitinib citrate tablet + Methotrexate tablet； group C was given Tofacitinib citrate tablet + Leflunomide tablet. Three groups were given relevant medicine for 6 months. Therapeutic efficacy and disease activity score 28 （DAS 28） of 3 groups as well as Sharp score， the levels of biochemical indicators [erythrocyte sedimentation rate （ESR）， C- reactive protein （CRP）]， immune indexes [rheumatoid factor （RF）， anti-cyclic peptide containing citrulline （anti-CCP） antibody]， serum cytokine indicators [interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α）] before and after treatment were observed； the occurrence of adverse drug reactions during treatment was recorded. RESULTS After treatment， the proportions of ACR50 and ACR70 patients in group A were significantly higher than groups B and C （P＜0.05）； DAS28 score， Sharp score， biochemical indicators， immune indexes and serum cytokine indicators of 3 groups were significantly lower than before treatment （P＜0.05）， and gradually decreased with prolonged treatment time； after 6 months of treatment， DAS28 score， Sharp score， RF， anti-CCP antibody， the levels of IL-6 and TNF-α in group A were significantly lower than group B and C （P＜0.05）. There was no significant difference in the incidence of diarrhea， nausea and vomiting， leukopenia， rash， abnormal liver and kidney function， or dizziness among 3 groups （P＞0.05）. CONCLUSIONS Tofacitinib combined with hydroxychloroquine shows good efficacy and safety for refractory RA.

Aim To investigate the effect of CDR132L(miR-132 antisense oligonucleotide)on vascular remode-ling and function in mice with hypertension and hyperlipidemia,and explore its possible mechanism.Methods A total of 30 8-week-old male C57BL/6 mice were randomly divided into three groups:control group,model group and CDR132L group,with 10 mice in each group.The control group received with a standard diet while the model group and CDR132L group received N-nitro-L-arginine methyl ester(L-NAME)and high-fat diet to induce hypertension and hyperlip-idemia.The CDR132L group was administered with intraperitoneal injection of CDR132L at a dose of 20 mg/kg once weekly for six consecutive weeks,whereas the control group and the model group were given intraperitoneal injection of an equivalent volume of normal saline.The tail-cuff method was utilized for blood pressure measurement,blood lipid and glucose levels were assayed by an automatic biochemical analyzer,the thoracic aorta structure was observed by HE staining,endothelium-dependent relaxation of the thoracic aorta was evaluated by the vascular ring test,the expression level of miR-132 in the thoracic aorta was measured by qPCR,the protein expression levels of Gab1 and endothelial nitric oxide synthase(eNOS)in the thoracic aorta were determined by Western blot.Results Compared with the control group,the model group demonstrated notable rises in systolic and diastolic blood pressure,serum triglyceride,total cholesterol lev-els,and body weight.Moreover,the intima of thoracic aorta and the thickness of vascular wall was uneven,the smooth muscle cells of the tunica media were arranged irregularly,with a large amount of fat deposition in the vascular wall,and the endothelium-dependent relaxation response of thoracic aorta was decreased(P＜0.05).The expression level of miR-132 in the thoracic aorta was significantly increased(P＜0.05),while the expression level of Gabl and eNOS protein was markedly decreased(P＜0.05).Compared with the model group,the CDR132L group showed no significant differences in systolic and diastolic blood pressure,serum triglyceride and total cholesterol levels,as well as body weight(P＞0.05).However,the CDR132L group exhibited a complete and smooth intima of the thoracic aorta with minimal intravascular lipid deposition,the thickness of the vascular wall was uniform,the smooth muscle cells of the tunica media were arranged order-ly,accompanied by enhanced endothelium-dependent relaxation response of the thoracic aorta(P＜0.05).The expression level of miR-132 in the thoracic aorta was significantly decreased(P＜0.05),while the expression levels of Gab1 and eNOS protein were significantly increased(P＜0.05).Conclusion CDR132L can improve vascular remodeling and endothelium-dependent relaxation in hypertensive and hyperlipidemia mice,which may be related to the decrease of miR-132 expression level and the up-regulation of Gab1 and eNOS protein expression levels in the thoracic aorta.

Objective To observe the effects of different concentrations of glucose on the proliferation and differentiation of primary osteoblasts.Methods The identification of mouse primary osteoblasts was performed by alkaline phosphatase (ALP)staining and Von Kossa staining.Treating osteoblasts with different dose of glucose (5.5,15.5,25.5 mmol/L),the osteoblasts proliferation,ALP staining,and Runx2,OB markers ALP and OCN mRNA expression were observed.Real-time PCR was used for the determination of Runx2,OB markers ALP and OCN mRNA expression.Results With the increasing glucose concentrations,the osteoblasts cell proliferation was decreased.Compared with 5.5 mmol/L normal glucose,the ALP staining in 15.5 mmol/L group and 25.5 mmol/L group were decreased.The expressions were decreased by (36.7±6.2)％ and (38.3±2.2)％ in Runx2 mRNA,(26.7±7.2)％ and (40.4±4.3)％ in OCN mRNA respectively.ALP in 15.5 mmol/L group was reduced by (33.3±10.2)％,but increased by(50.8±10.4) ％ in 15.5 mmol/L group.Conclusions High glucose may decrease osteoblasts proliferation and activity,which may be one of the key pathogenesis factors of diabetic osteoporosis.

Objective To investigate the effect of 620 nm red light on chondrogenic differentiation in rat precartilaginous stem cells (PSCs). Methods Rats' PSCs were isolated and purified using magnetically activated cell sorting and cultured in vitro.The PSCs were exposed once to 620 nm wavelength red light from a light-emitting diode (LED) with an irradiation energy of 0.5 J/cm2,1 J/cm2,2 J/cm2 or 4 J/cm2.Any effect was confirmed by Alcian blue staining,immunohistochemistry and observing histomorphological changes under a light microscope,as well as detection using a reverse transcription polymerase chain reaction (RT-PCR). Results After being induced for 14 d,the PSCs exhibited polygonal and round shapes. Alcian blue and type Ⅱ collagen immunohistoehemistry staining showed positive results,but the control group had no significant change.RT-PCR showed that the mRNA expression of Sox9 and type Ⅱ collagen increased significantly compared with the control group. Conclusion Low energy 620 nm red light can enhance chondrogenic differentiation in PSCs significantly.

The effects of high-intensity pulsed electromagnetic stimulation (HIPEMS) on proliferation and differentiation of neonatal rat neural stem cells in vitro were investigated. Neural stem cells derived from neonatal rats were exposed to 0.1 Hz,0.5-10 Tesla (T) [8 groups of B-I,respectively],5 stimuli of HIPEMF. The sham exposure controls were correspondingly established. Inverted phase contrast microscope was used to observe the cultured cells,MTT assay to detect the viability of the cells as expressed by absorhance (A) value,and flow cytometry to measure differentiation of neural stem cells. The results showed that A values of neural stem cells in both 3.0 T and 4.0 T groups were significantly higher than the other groups 24 to 168 h post HPEMS,indicating a strong promotion of the growth of neural stem cells (P＜0.05). The A values of neural stem cells in the 6.0 T,8.0 T,and 10.0 T groups were lower than the sham exposure control group,indicating a restraint of the growth of neural stem cells. The rate of neuron-specific enolase-positive neurons revealed by flow cytometry in HPEMS groups was the same as that in control group (P＞0.05). It was suggested that 0.1 Hz,5 pulses stimulation of HPEMS within certain scale of intensity (0.5-10.0 T),significantly promoted the growth of neural stem cells with the rational intensity being 4.0 T.

The effects of high-intensity pulsed electromagnetic stimulation (HIPEMS) on proliferation and differentiation of neonatal rat neural stem cells in vitro were investigated. Neural stem cells derived from neonatal rats were exposed to 0.1 Hz, 0.5-10 Tesla (T) [8 groups of B-I, respectively], 5 stimuli of HIPEMF. The sham exposure controls were correspondingly established. Inverted phase contrast microscope was used to observe the cultured cells, MTT assay to detect the viability of the cells as expressed by absorbance (A) value, and flow cytometry to measure differentiation of neural stem cells. The results showed that A values of neural stem cells in both 3.0 T and 4.0 T groups were significantly higher than the other groups 24 to 168 h post HPEMS, indicating a strong promotion of the growth of neural stem cells (P<0.05). The A values of neural stem cells in the 6.0 T, 8.0 T, and 10.0 T groups were lower than the sham exposure control group, indicating a restraint of the growth of neural stem cells. The rate of neuron-specific enolase-positive neurons revealed by flow cytometry in HPEMS groups was the same as that in control group (P>0.05). It was suggested that 0.1 Hz, 5 pulses stimulation of HPEMS within certain scale of intensity (0.5-10.0 T), significantly promoted the growth of neural stem cells with the rational intensity being 4.0 T.

BACKGROUND: Neuronal apoptosis produced a main effect in secondary injury after injury of central nervous system. Recently, minocycline has been reported to protect neurologic function evidently by decreasing apoptosis.OBJECTIVE:To study the effect of minocycline on apoptosis and expression of related factor, and protective effect of minocycline on neurologic function of injured spine after spinal cord injury (SCI) in rats.DESIGN: A randomized controlled animal study.SETTING: Tongji Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology.MATERIALS: The experiment was performed at the Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology between November 2003 and December 2004. A total of 36male SD rats were divided into four groups: ①Blank control group (group A) with 6 rats, ②injured group (group B) with 10 rats, ③minocycline treatment group (group C) with 10 rats, and ④methylprednisolone treatment group (group D) with 10 rats.METHODS: The animal SCI model was established. Spinal cords of rats in the group A were exposed without injury. Spines of rats in the group B were injured without treatment. Rats of group C was treated with minocycline (50 mg/kg) by intraperitoneal injection at 1 hour after injury, and 24 hours later another 50 mg/kg was given, and then another 25 mg/kg was injected for 5 days. Methylprednisolone (100 mg/kg) was intraperitoneally injected 1 hour after injury in the group D. The rats in the group A and the group B were treated with saline, and the method of administration was the same with that of the group C.MAIN OUTCOME MEASURES: ①Neurofunction evaluation of rats in each group. ②Expressions of Bcl-2 family members (Bcl-2, Bcl-xl, and Bax) were tested with immunocytochemical method. ③Apoptosis index of spinal tissue of rats of each group.RESULTS: A total of 36 rats were involved in the result analysis. ①Neurofunction of rats in the group C increased as compared with the group B (P ＜ 0.05), and there was insignificant difference between group C and the group D (P ＞ 0.05). ②Bcl-2 positive expressive cells in the group C was more than that in the group B, and showed obvious difference between the two groups [(62.53±7.76)%, (35.14±3.70)%,P ＜ 0.05]. There was insignificant difference between expressions of Bcl-xl and Bax in group C as compared with the group B (P ＞ 0.05). ③The number of apoptosis positive cells in the group B was more than that in the group C and D [(44.63± 5.90)%, (32.71± 5.26)%, (34.31± 6.84)%,P ＜ 0.05].CONCLUSION: Minocycline can up-regulate the expression of Bcl-2, and raise the ratio of Bcl-2/Bax, so as to inhibit the neural apoptosis of spine,which have protective effect on neurologic function of injured spine.

In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45-0.11 in MG63/DN cells, while 3.40+/-0.12 in the cells transfected with blank vector (MG63/I), (P<0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P<0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.

In order to study the effects of dominant negative human telomerase reverse transcriptase (DN-hTERT) on cell growth and telomerase activity in osteosarcoma cell line MG63, MG63 cells were transfected with DN-hTERT-IRES2-EGFP9 (DN) or IRES2-EGF (I, blank vector) with lipofectamine 2000. The stably transfected cells were selected with G-418. Cell growth properties were examined under a fluorescence microscope. The hTERT mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). Telomerase activities were measured by TRAP-ELISE. The tumorigenicity was studied with tumor xenografts by subcutaneous injection of cancer cells into nude mice. The results showed that cell growth was suppressed in MG63 cells transfected with DN-hTERT. The hTERT mRNA was increased in N-hTERT transfected-MG63 cells (MG63/DN). The telomerase activity was 2.45±0.11 in MG63/DN cells, while 3.40±0.12 in the cells transfected with blank vector (MG63/I), (P＜0.05); DN-hTERT-expressing clones did not form tumors in 2 weeks, but the ratio of tumorigenesis was 30 % in nude mice bearing MG63/I (P＜0.01). It was concluded that DN-hTERT could specifically inhibit the cell growth and telomerase activity in MG63 cells.

ObjectiveTo explore the early effects of transplantation of fetal spinal cord (FSC) and basic fibroblast growth factor (bFGF) on calcium, magnesium of injured spinal cord.MethodsThe spinal cord injury (SCI) animal model was established with Wistar rats. The rats were divided into 3 groups. The group A was received FSC cells suspension 5 ml after injury. The group B was received FSC cells suspension and bFGF 20 μl. The group C was not treated with any therapy. The values of Ca2+, Mg2+ of injured spinal cord tissues were measured at 6, 24 hours after injury. The morphyological results were observed by electron microscope on the fifth day.ResultsThe degree of ionic disorder in FSC+bFGF treated rats was strikingly ameliorated and the contents of H2O also decreased. The morphyological finding revealed that in FSC+bFGF treated rats, the damage of gray and white matters was slight in contrast to those of control group rats, which appeared a typically damage.ConclusionTransplantation of FSC and bFGF has some protective effects on contused spinal cord in rats.