AIM: To investigate the feasibility and infection efficiency of MSCs as the target cells of gene delivery mediated by adenoviral vector carrying CTLA4Ig gene, and to study the mechanism of transgenic MSC to inhibit immune response ex vivo. METHODS: The recombinant adenovirus containing CTLA4Ig gene was constructed, by which rat MSCs with various multiplicity of infection (MOI) were conducted. The infection efficiency was analyzed with FACS and fluorescence microscope. The expression of CTLA4Ig protein in transgenic MSCs was detected by FACS and western blot. Co-culturing the transgenic MSCs with mixed lymphocytes, the inhibitory effect of transgenic MSCs on lymphocyte proliferation was also observed. RESULTS: The adenoviral vector delivered CTLA4Ig gene with high efficiency to MSCs. The expression of CTLA4Ig protein was detected in transgenic MSCs. The gene modified MSCs inhibited the proliferation of mixed lymphocytes and maximal inhibition rate was observed on day 4 of MLR. The inhibition induced by CTLA4Ig was donor-specific. CONCLUSION: MSCs is a promising target cell for gene delivery. The expressed CTLA4Ig specifically inhibits the lymphocyte proliferation ex vivo.

Objective To investigate the effect of running exercise on cartilage in rats with an unstable knee joint.Methods Twenty 8-week-old Sprague-Dawley rats had their left anterior cruciate ligament cut to model an unstable knee.They were randomly divided into a control group and an experimental group,10 rats in each group.The control group was given no intervention,while the experimental group accepted running exercise training on na animal treadmill at a velocity of 15 m/min for an hour every day.After 3 and 6 weeks of training,5 rats were sacrificed and cartilage from the medial condyle of the femur was sampled,decalcificated,embedded and sliced on the sagittal plane.After hematoxylin-eosin staining,toluidine blue staining and immunohistochemical staining,the cartilage thickness,Mankin' score,the content of matrix collagen and the proteoglycan content of the cartilage matrix were assessed,and the shape and structure of the unstable knee joints were observed under a transmission electron microscope.Results The cartilage thicknesses and Mankin's scores at 6 weeks were significantly different from those at 3 weeks in both groups.In the experimental group the average thickness of cartilage was 154 ± 13 μm at 3 weeks and 131 ± 15 μm at 6 weeks.The corresponding Mankin's scores were 9.93 ± 1.36 and 11.23 ± 1.57,respectively.Both were significantly different from the control group averages at the same time points.There was also a significant difference in the positive rate of toluidine blue and collagen type Ⅱ staining between the experimental group and the control group at both time points,and in the experimental group between 3 and 6 weeks of training.After 3 weeks of training,fewer chondrocytes were observed under the transmission electron microscope in the experimental group,and fissures were seen on the surface of the cartilages.However,3 weeks later,quite a few ruptures and a lot of necrotic cells could be seen.Conclusions Running exercise can damage the cartilage of unstable knee joints and speed up the development of osteoarthritis.Even moderate exercise could aggravate damage to unstable joints and the cartilage matrix,and accelerate chondrocyte degeneration.

OBJECTIVE:To establish a method for the determination of 4 flavonoids constituents in Yinqiao capsules. METH-ODS:HPLC method was adopted. The Hypersil ODS C18 column was used with the mobile phase A of methanol-water-acetic acid (10∶88∶2,V/V/V)and B of methanol-water-acetic acid(88∶10∶2,V/V/V)in gradient elution at the flow rate of 1.0 ml/min;the de-tection wavelength was 327 nm,the column temperature was 25 ℃,and the volume was 10 μl. RESULTS:There was a good linear relationship between the amount of quercetin and peak area in the range of 0.050 9-1.018 0 μg(r=0.999 8),kaempferide in the range of 0.050 2-1.004 0 μg(r=0.999 5),isorhamnetin in the range of 0.051 0-1.020 0 μg(r=0.999 4)and rutin in the range of 0.050 4-1.007 0 μg(r=0.999 8). RSDs of precision,stability and repeatability tests were <2%. The average recoveries were 100.09%(RSD=0.93%,n=9),99.83%(RSD=0.75%,n=9),100.51%(RSD=1.17%,n=9) and 101.19%(RSD=1.08%,n=9). CONCLUSIONS:The method is amount specific,stable and reproducible and can be used for the quality control of Yinqiao capsules.

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Objective To evaluate how proprioception affect ankle stability through comparing angle position awareness and peroneus reaction time between chronic ankle lateral instability patients and healthy controls.Methods A total of 51 participants were recruited into an experimental group of 21 patients with chronic ankle lateral instability (17 males,aged 31.6±2.6) and a control group of 30 healthy counterparts (24 males,aged 34.2±2.3).All the participants were asked to reoccur passive ankle position under the angular velocity at 2 degree per second when they were resting with non-weight bearing in their recruited ankles.The muscle reaction time (MRT)of peroneus longus(PL) and peroneus brevis (PB) in all the recruited ankles was measured during sudden ankle inversion both with and without ankle protective brace wearing.Results The difference between angle recurrence and the target angle (ankle inversion 20° and 30°) was significantly higher (P＜0.05) in the experimental group compared to the control group.The average MRTs of PL and PB were also significantly longer (P＜0.05) in the experimental group than the control group,whether wearing ankle protective braces or not.However,within both groups,no significant differences of PL and PB's MRT were identified between brace wearing and no brace (P＞0.05).Conclusions In patients with chronic lateral ankle instability,the position awareness decreases and the reaction time of peroneus is prolonged.Ankle braces can provide mechanical protection to the injured joints,but cannot promote MRT significantly.

Objective To discuss clinical,electroencephalogram(EEG) and PRRT2 gene mutation by reporting a febrile seizure (FS) with paroxysmal kinesigenic dyskinesia (PKD) family.Methods Detailed clinical data of the family were collected.The proband (Ⅳ1) and another 4 patients (Ⅲ1,Ⅲ4,Ⅳ2,Ⅳ3)were studied through clinical examinations.Clinical symptoms of Ⅳ2 were not typical,who was diagnosed as a suspected case.Mutation analysis of PRRT2 gene was screened by polymerase chain reaction (PCR) and DNA direct sequencing in 5 patients (Ⅳ1,Ⅲ1,Ⅲ4,Ⅳ2,Ⅳ3) and 4 unaffected family members (Ⅱ2,Ⅲ2,Ⅲ5,Ⅳ4).Results PKD patients had brief involuntary movements in the limbs or trunk induced by sudden voluntary movement when patients were in the stationary state since the teenagers.Two cases (Ⅲ,Ⅲ4) were accompanied by FS.Three cases(Ⅳ1,Ⅲ1 and Ⅲ4)had abnormal EEG records.The PRRT2 gene mutation (c.649dupC mutation) was identified in a healthy member (Ⅳ4) and 4 patients (Ⅳ1,Ⅲ1,Ⅲ4,Ⅳ3).Conclusions FS with PKD family has a PRRT2 gene mutation.The diagnosis is mainly based on family history,typical clinical manifestations and genetic test.This kind of disease may have pre-symptomatic patients.

Objective To determine the feasibility of magnetically labeling and tracking mesenchymal stem cells(MSCs)in vitro by using a gadolinium and fluorescent bi-functionally transfection agent of polyethylenimine.Methods A gadolinium bifunctional transfection reagent complex was obtained after the linear polyethylenimine derivative(JetPEI-FluoR)was incubated with Gd-DTPA.Mesenchymal stem cells isolated from the bone marrows of SD rats were cultured and expanded.The mesenchymal stem cells were incubated with the bi-functional labeling agents.After labeling,the MSCs were examined with fluoroscope and electron microscope and the biological characters were detected including trypan blue exclusion test,MTT,and apoptosis detection.On a 1.5 T MR system,the labeled MSCs were examined with spin echo T1 WI and T2 WI and T1 measurement with mixed sequence.After labeling,the cells were cultured and undergone routine passage.Prior MR examinations were repeated for each passage of labeled cells.All data was statistically prolessed with SPSS for Windows.Results Of 5×105 MSCs incubated with the bi-functional agents,4.25×105 MSCs were successfully labeled,the percentage of labeled MSCs was 85% fluoroscopically.The high density electron particles of gadolinium observed electron microscopically existed around cellular apparatuses,especially around Golgi apparatus.In trypan blue exclusion test,the exclusion rate of labeled MSCs with incubation duration of 3,6,12,24 h was(96.55±2.90)%,(94.17±2.56)%,(97.16±3.12)% and(94.23±2.67)%,respectively.The corresponding exclusion rate of unlabeled MSCs was(95.86±2.67)%,(92.04±2.21)%,(93.38±3.64)%and(92.12±2.53)%,respectively.There was no statistical difference of trypan blue exclusion rate between labeled cells and control unlabeled cells within 24 hours of incubation(F=4.523,P＞0.05).In the proliferation test,the optical absorption value of labeled MSC with 2.5,5.0,10.0,20.0,30.0 and 40.0 μl bi-functional labeling agent was(0.1884±0.0151),(0.1878±0.0190),(0.1741±0.0160),(0.1135±0.0215),(0.1079±0.0145)and(0.0811±0.0079),respectively.The corresponding optical absorption value of unlabeled MSCs was(0.1940±0.0116).The optical absorption value of labeled cells was not affected in case of less than 30.0 μl of Gd-DTPA(q'=0.2225-0.9458,P＞0.05).The apoptosis index for labeled cells and unlabeled cells were 5.08% and 3.86%,respectively.On T1 WI,the signal intensity and T1 relaxation time of unlabeled cells and labeled cells were 240.3±24.7 and(2457±56)ms,336.2±20.7 and(1102±64)ms,respectively,and there were significant statistical difference(t=12.656,17.889,P＜0.01).The minimal amount of cells which was detectable for T1 WI was 5×103.After routine passage,the gadolinium in the cells gradually decreased and could be tracked by MRI until the fifth passage.Conclusions The gadolinium and fluorescent bi-functionally labeling rat bone marrow mesenchymal stem cell by using the transfection agent of polyethylenimine is feasible,efficient and safe.The labeled cells could be tracked in vitro on MR imaging.

For most stage T1 renal tumors, it is still necessary to block the renal pedicle to achieve the purpose of resection.However, it is not conducive to the recovery of postoperative renal function.Therefore, laser plays an important role in partial nephrectomy in order to achieve zero ischemia, and the aim of this review is to explain the research progress of laser in partial nephrectomy.

Gene therapy has been considered as a promising method for treatment of many diseases, such as acquired and genetic diseases. At present, there are two major vehicles for gene delivery including viral vectors and nonviral vectors. Viral vectors appear as high gene transfection efficiency, but some deficiencies such as inflammatory responses, recombination and mutagenesis have limited their use. On account of low pathogenicity, safety and cost-effectiveness, nonviral vectors have been attracted much attention. Cationic polymers are one of the nonviral vectors which have been widely studied. This review focuses on the structure of the cationic polymers and the interaction mechanism between the vector and DNA. We try to provide a framework for the future design and synthesis of nonviral vectors with high transfection efficiency and low toxicity for gene therapy.
Cations ; chemistry ; DNA ; genetics ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Polymers ; chemistry

The incidence of stroke increases year by year.It seriously affects the quality of life in patients.The pathogenesis of stroke is related to a variety of factors,involving genetic polymorphisms,biochemical mechanisms,and inflammatory effect.Among them,vascular endothelial growth factor (VEGF) has become one of the hotspots of research on the pathogenesis of stroke in recent years.This article reviews the correlation between VEGF gene polymorphism and stroke.