Knee Osteoarthritis is one of the most common degenerative diseases.Although the morbidity of knee osteoarthritis increases dramatically,there is no effective medication that can repair the damaged articular cartilage and restore the joint function.Recently,with the technical development of cell transplantation and regulation,more attentions have been paid in this area.The techniques included osteochondral autograft transplantation,mosaicplasty,chondrocytes based matrix-induced autologous chondrocyte implantation,and mesenchyma stem cells based cell therapy.The study of cell differentiation is also more in-depth.It has been confirmed that all of these methods can alleviate the symptoms and improve the function of knee osteoarthritis to some extent.Stem cell therapy seems have greater advantage than other intervention.Massive chondrocytes can be gained through induction and culture of mesenchyma stem cells in vitro,and regenerated cartilage could be found on the surface after delivering stem cells into knee joint through a certain way.These technologies are hopeful to prevent from knee osteoarthritis or even reverse the progress of it at early stage.However,there are a variety of problems about in vitro cell transplantation,such as the unknowing regulation of mesenchyma stem cells,the difficulty of keeping the characteristic stability of new born chondrocytes,the integrity of new cartilage tissue and the way chondrocytes delivered.Cell therapy has been applied both in basic research and in clinic trials.The present review is focused in the current progress of cell therapy in knee osteoarthritis and discuss the challenges and troubles.Furthermore,we also summarize a series of ongoing clinical trials,and try to find out the exact clinical effects of cartilage repair by using cell therapy.

Objective:To explore the application of real-time transrectal ultrasound (TRUS) during seminal vesiculoscopy in infertile men with azoospermia or oligoasthenospermia.Methods:We retrospectively analyzed the clinical data of 25 cases of azoospermia or oligoasthenospermia due to ejaculate ducts obstruction who were treated with real-time transrectal ultrasound-guided seminal vesiculoscopy between September 2011 and December 2015. Patients’ age was(29.4±4.5) years. All patients accepted semen analysis, serum sex hormone, MRI, TRUS and then diagnosed as obstructive azoospermia, and 13 cases had intractable obstructive azoospermia or oligoasthenospermia after the failure of simple seminal vesiculoscopy(the path to the ejaculatory duct and seminal vesicle couldn’t be found). All patients were treated with seminal vesiculoscopy under real-time guidance with TRUS. We assessed the success rate of surgery, surgical time and complications.Results:The scope was successfully inserted into the seminal vesicle in 21 of the 25 cases (success rate, 84%). The median operative time was 75(31, 148) min. None of the patients developed severe complications. Among 4 failure cases (4/25, 16%), 1 was due to abnormal congenital development. In 2 cases, a clear outlet of the dual ejaculatory duct could not be found after it was inserted into the prostatic utricle. One case was considered as a Müllerian tubular cyst, and the seminal vesicle scope was used to assess the cystic side wall. The 21 patients were followed up for 3 to 6 months, semen volume 2.0(0-5.2)ml, total sperm 28(0-832) ×10 6/ejaculate, sperm density 5.6(0-110.3)×10 6/ml, mobility rate of sperm 5.4%(0-63.6%), and the differences were significant as compared to that before the surgery [semen volume 0.4(0-2.8)ml, total sperm 0(0-342)×10 6/ejaculate, sperm density 0(0-90.7)×10 6/ml, mobility rate of sperm 0(0-24.1%), all P<0.05]. Among the 17 patients who underwent follow-up of 5 to 9 years, 3 patients was conceived naturally and 9 patients’ postoperative sperm quality has improved and pregnancy in vitro fertilization by extracting sperm from semen. Conclusions:Intraoperative real-time transrectal ultrasound guidance can improved the success rate of seminal vesiculoscopy and promoted operative safety.

To begin with, we constructed cfp10-esat6 fusion gene and its prokaryotic expression vector and had it express in E. coli. By GeneSOEing techniques, a fusion gene was constructed by splicing cfpl0 gene and esat6 gene, and then was cloned into pGEX-4T-1 plasmid. Secondly, we constructed the prokaryotic expression recombinant plasmid pGcfp10-esat6. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis, The E. coli BL21 containing the recombinant plasmid was induced by IPTG (Isopropy-beta-D-thiogalatoside). The fusion protein CFP10-ESAT6 with GST-tag about 42 kDa was expressed and purified with GST-fusion protein purification kit,The expression of cfp10-esat6 fusion gene was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The sequence of cfp10 and esat6 in recombinant plasmid was consistent with that of GenBank report. The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E. coil. The fusion protein was purified and the purity reached 90%. Its antigenicity was confirmed by Western-blotting. The prokaryotic expression vector (pGcfp1o-esat6) was constructed successfully, and the fusion protein CFP10-ESAT6 was obtained. This study provided an experimental basis for potential application of the recombinant CFP10-ESAT6 in the diagnosis of tuberculosis.
Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Objective: To evaluate the efficacy and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in prophylaxis neutropenia after chemotherapy in patients with lymphoma. Methods: This was a multicenter, single arm, open, phase Ⅳ clinical trial. Included 410 patients with lymphoma received multiple cycles of chemotherapy and PEG-rhG-CSF was administrated as prophylactic. The primary endpoint was the incidence of Ⅲ/Ⅳ grade neutropenia and febrile neutropenia (FN) after each chemotherapy cycle. Meanwhile the rate of antibiotics application during the whole period of chemotherapy was observed. Results: ①Among the 410 patients, 8 cases (1.95%) were contrary to the selected criteria, 35 cases (8.54%) lost, 19 cases (4.63%) experienced adverse events, 12 cases (2.93%) were eligible for the termination criteria, 15 cases (3.66%) develpoed disease progression or recurrence, thus the rest 321 cases (78.29%) were into the Per Protocol Set. ②During the first to fourth treatment cycles, the incidences of grade Ⅳ neutropenia after prophylactic use of PEG-rhG-CSF were 19.14% (49/256) , 12.5% (32/256) , 12.18% (24/197) , 13.61% (20/147) , respectively. The incidences of FN were 3.52% (9/256) , 0.39% (1/256) , 2.54% (5/197) , 2.04% (3/147) , respectively. After secondary prophylactic use of PEG-rhG-CSF, the incidences of Ⅳ grade neutropenia decreased from 61.54% (40/65) in the screening cycle to 16.92% (11/65) , 18.46% (12/65) and 20.75% (11/53) in 1-3 cycles, respectively. The incidences of FN decreased from 16.92% (11/65) in the screening cycle to 1.54% (1/65) , 4.62% (3/65) , 3.77% (2/53) in 1-3 cycles, respectively. ③The proportion of patients who received antibiotic therapy during the whole period of chemotherapy was 34.39% (141/410) . ④The incidence of adverse events associated with PEG-rhG-CSF was 4.63% (19/410) . The most common adverse events were bone pain[3.90% (16/410) ], fatigue (0.49%) and fever (0.24%) . Conclusion During the chemotherapy in patients with lymphoma, the prophylactic use of PEG-rhG-CSF could effectively reduce the incidences of grade Ⅲ/Ⅳ neutropenia and FN, which ensures that patients with lymphoma receive standard-dose chemotherapy to improve its cure rate.

BACKGROUND:Heterotopic ossification of skeletal muscle is a clinically serious complication.For heterotopic ossification of skeletal muscles,the cells involved in the process of heterotopic ossification remain unclear. OBJECTIVE:To investigate the involvement of myocytes,fascia cells,and endothelial cells in the process of heterotopic ossification in skeletal muscle and to observe the cell origin of heterotopic ossification in skeletal muscle induced by bone morphogenetic protein 4. METHODS:Both C2C12 cells and the myotubes formed by the C2C12 cells in the induction medium were cultured,and 500 ng/mL bone morphogenetic protein 4 was added to the medium respectively,and whether the C2C12 cells and myotubes continued to proliferate within 10 days under the treatment were observed under a microscope.Myogenic cells(L6,derived from rats)and fibroblast-derived cells(derived from human)were co-cultured.After treatment with 500 ng/mL bone morphogenetic protein 4 and 10 ng/mL transforming growth factor-β,osteogenic and chondrogenic differentiation potential within 21 days were observed using Safranine O staining and Alcian blue staining.Using transgenic animal FVB/N-TgN(TIE2-LacZ)182Sato mice,15 μL of adeno-associated virus-bone morphogenetic protein 4(5×1010 PFU/mL)were implanted in the thigh muscle space of genetic mice for 10 and 14 days.X-gal staining was used to observe the formation of new blood vessel endothelium in the differentiated bone. RESULTS AND CONCLUSION:(1)Bone morphogenetic protein 4 caused myotube breakdown and increased C2C12 cell proliferation.Compared with other groups,the pure fibroblast-derived cell group had a higher area of positive alcian blue and safarin O staining(P<0.05)and a lower area of alkaline phosphatase staining(P<0.05),while the pure L6 group had a bigger area of alkaline phosphatase staining(P<0.05)but a smaller area of positive alcian blue and safarin O staining(P<0.05).(2)Transplantation of adeno-associated virus-bone morphogenetic protein 4-adsorbed gelatin sponge into FVB/N-TgN(TIE2-LacZ)182Sato mice resulted in heterotopic ossification.(3)X-gal staining results demonstrated that there was no obvious staining in chondrocytes and differentiated bones and Tie2+ endothelial cells did not participate in the formation of the alienated bone.(4)These findings verify that fibroblasts are the primary source of osteoblasts during the adeno-associated virus-bone morphogenetic protein 4-induced ectopic endochondral ossification in skeletal muscle,but myogenic cells are the main source of osteoblasts.Tie2+ endothelial cells might not be the cell source for cartilage and bone.