BACKGROUND:Incidence of diabetic lower extremity vascular disease is increasing, so how to improve blood vessels of the lower limbs and increase angiogenesis becomes the research focus. Umbilical cord mesenchymal stem cells have been employed clinical y via local intramuscular injection, but the specific therapeutic effect and mechanism are not clear.
OBJECTIVE:To investigate the effects of hypoxic preconditioning and cobalt chloride medium on the differentiation of umbilical cord mesenchymal stem cells into vascular endothelial-like cells in vitro.
METHODS:Umbilical cord mesenchymal stem cells were isolated and cultured, and then treated with different concentrations of cobalt chloride. Enzyme linked immunosorbent assay was used to detect levels of basic fibroblast growth factor and vascular endothelial growth factor gene in cellsupernatants, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to detect cellproliferation. The safety of umbilical cord mesenchymal stem cells before and after cobalt chloride induction was detected using chromosome. The umbilical cord mesenchymal stem cells were cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10μg/L vascular endothelial growth factor, 10μg/L basic fibroblast growth factor and 10%fetal bovine serum, which were induced to differentiate into vascular endothelial-like cells. Endothelial-like cellphenotype CD31 and von Wil ebrand factor were identified before and after induction, through the observation of three-dimensional model of angiogenesis, the angiogenic capacity of umbilical cord mesenchymal stem cells was determined.
RESULTS AND CONCLUSION:Umbilical cord mesenchymal stem cells strongly expressed the surface marks. After the cobalt chloride induction, the proliferation of umbilical cord mesenchymal stem cells was positivelycorrelated with the time of induction. Based on the levels of vascular endothelial growth factor and basic fibroblast growth factor, the optimal concentration of cobalt chloride was 200 μmol/L. Chromosome detection showed the stability of cells after cobalt chloride induction. After induction, CD31 and von Wil ebrand factor were strongly expressed. Three-dimensional observation showed umbilical cord mesenchymal stem cells could be induced to form the lumen-like structure with different diameter sizes, and umbilical cord mesenchymal stem cells could be induced to differentiate into endothelial-like cells, and have a angiogenic capacity.
RESULTS AND CONCLUSION:Umbilical cord mesenchymal stem cells strongly expressed the surface marks. After the cobalt chloride induction, the proliferation of umbilical cord mesenchymal stem cells was positivelycorrelated with the time of induction. Based on the levels of vascular endothelial growth factor and basic fibroblast growth factor, the optimal concentration of cobalt chloride was 200 μmol/L. Chromosome detection showed the stability of cells after cobalt chloride induction. After induction, CD31 and von Wil ebrand factor were strongly expressed. Three-dimensional observation showed umbilical cord mesenchymal stem cells could be induced to form the lumen-like structure with different diameter sizes, and umbilical cord mesenchymal stem cells could be induced to differentiate into endothelial-like cells, and have a angiogenic capacity.

Background Implantable contact lens (ICL) surgery is a primary intraocular refractive corrective surgery for high myopia.However,whether there will be enough distance between ICL and anterior face of lens to avoid the occurrence of anterior capsular cataract in non-accommodated and the largest physiological accommodated state after ICL implantation is worthy of investigation.Objective The purpose of this study was to investigate the alteration of lens vault after ICL implantation and explore the relationship between accommodation and change of lens vault.Methods A observational study was carried out.Sixty-six eyes of 33 patients with high myopia who received ICL implantation were enrolled in Affiliated First Hospital of Guiyang Medical College from May to November,2012.Best corrected visual acuity (BCVA),uncorrected visual acuity (UCVA),refractive diopter were regularly examined using synthetical optometry,and crystalline lens rise (CLR) and lens vault in non-accommodative or accommodative condition were observed by the anterior segment OCT (Visante OCT) and ultrasound biomicoscopy (UBM) before operation and 1 day,1 week,l month and 3 months after operation.Data were analyzed with SPSS version 16.0.Repeated measurement one-way analysis of variance was used to compare the differences of vision and refractive diopter among various time points.The relationship between accommodation and CLR was assessed using Pearson linear correlation.The alteration of CLR with accommodation change was analyzed by linear regression equation.Lens vault was measured and compared between non-accommodation and maximal physical accommodation status by paired t test.Results The postoperative UCVA was improved in comparison with preoperative BCVA,and the postoperative diopter was significantly lower than that of preoperation,with significant differences among various time points (F =16.904,P =0.000 ; F =1.498,P =0.000),and the diopter was stable after operation.A positive correlation was found between the alteration of CLR and accommodation under the physical accommodative stimulation in high myopic eyes (R2 =0.49,P =0.00).Under physiological accommodation,CLR elevated 20 μm for per 1.0 D accommodation.In addition,the difference of lens vault values within postoperative 3 months was statistically significant (F=16.025,P=0.000).The lens vault values lowed with the enlargement of accommodation in 48.5％ eyes,and the lens vault values increased with the enlargement of accommodation in 50.0％ eyes.However,1.5％ of the lens vault were in a stable state under the maximal physiological accommodated condition 3 months after operation.Lens vault were greater than zero in 97.0％ eyes (64/66),and only 3.0％ eyes (2/66) were zero under the maximalphysiological accommodated condition.Significant differences were seen in the lens vault between nonaccommodated and the maximal physiological accommodated state 1 day or 1 week after operation (t =4.755,P =0.000 ;t =3.327,P =0.001) ; but there was no statistical significance in 1 month or 3 months after operation (t =1.544,P=0.127,t=0.621,P=0.537).Conclusions During physiological accommodation,the alteration of CLR with accommodation in high myopic eyes.The location of ICL in the eyes is unstable within 3 months after operation.Majority of operative eyes remain enough vault in the maximal physiological accommodated state,but minority of operative eyes occur contact of ICL with the anterior surface of lens.Whether this contact causes anterior capsular cataract still needs to study.

Objective To observe the effects of secreted protein,acidic and rich in cysteine (SPARC) on the expression of TGF-β1 and collagen type Ⅰ in cultured human keloid fibroblasts by real-time fluorescence quantitative RT-PCR.Methods In vitro keloid fibroblasts were stimulated by different concentrations of recombinant human SPARC,and with the control group for comparison,real-time fluorescence quantitative RT-PCR to detect expression of TGFβ1 and collagen type Ⅰ.Results Compared with the control group,the expression of TGF-β1 and collagen type Ⅰ was significantly increased in the experimental group.Conclusions SPARC could enhance the expression of TGF-β1 and collagen type Ⅰ in keloid fibroblasts significantly.

BACKGROUND:Studies have shown that the number of osteoblasts is often decreased after osteoporosis, and osteoblast replacement therapy becomes a new target for the treatment of osteoporosis. OBJECTIVE:To observe the osteogenic differentiation of human bone marrow mesenchymal stem cels cultured in dexamethasone, vitamin C and beta-glycerophosphate. METHODS:Mesenchymal stem cels were isolated and purified from adult bone marrow using human lymphocyte separation medium. The expression of cel surface markers was detected by flow cytometry. Cel ultrastructure was observed by transmission electron microscope. Then, the bone marrow mesenchymal stem cels were cultured in osteogenic induction medium containing dexamethasone, vitamin C andβ-glycerophosphate, and RT-PCR was used to detect the bone morphogenetic protein-2 mRNA expression after osteogenic induction. RESULTS AND CONCLUSION:A large number of adherent cels were visible as fibrous growth at 2 weeks after culture and strongly expressed CD44, CD29, but did not express CD34, CD45. These cels could be induced to differentiate into osteoblasts, and express bone morphogenetic protein-2 mRNA. Alizarin red staining and alkaline phosphatase staining were positive for the cels. These findings suggest that human bone marrow mesenchymal stem cels cultured in dexamethasone, vitamin C and beta-glycerophosphate can differentiate into osteoblasts, and has a potential for the treatment of osteoporosis.

BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.

BACKGROUND:Stem cels are induced to differentiate into endothelial-like cels that can be used for the treatment of diabetic lower extremity vascular disease. However, it is unclear whether these endothelial-like cels can completely replace endothelial cels to improve vascular disease and what are the differences between endothelial-like cels and endothelial cels. OBJECTIVE:To explore the differences and similarities between endothelial-like cels and human umbilical vein endothelial cels in the aspects of morphology, function, and viability. METHODS:Umbilical cord mesenchymal stem cels and umbilical vein endothelial cels were isolated, cultured and identified using flow cytometry and immunohistochemical method. Isolated umbilical cord mesenchymal stem cels were induced in DMEM-LG/F12 containing 10 μg/L vascular endothelial growth factor, 10 μg/L basic
fibroblast growth factor and 2% fetal bovine serum to differentiate into endothelial-like cels folowed by immunohistochemical identification. To compare endothelial-like cels with human umbilical vein endothelial cels, cel migration detection, active substance measurement and three-dimensional angiogenesis test were performed. RESULTS AND CONCLUSION:Isolated umbilical cord mesenchymal stem cels strongly expressed the surface markers of mesenchymal stem cels, and human umbilical vein endothelial cels strongly expressed CD31 and VWF. After induction, the umbilical cord mesenchymal stem cels were identified to highly express CD31 and VWF. Through cel migration, active substance and three-dimensional angiogenesis tests, endothelial-like cels were similar to endothelial cels in the function and activity, and superior to endothelial cels. Cite this article:Hao XJ, Hao HY, Zhu MJ, Yuan Z, Li WW, Chen J, Zhu LY. Endothelial-like cels versus human umbilical vein endothelial cels. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):83-88.

Objective To study the presence of CD4+CD25+regulatory T cells and hepatitis B virus(HBV)specific cytotoxie T lymphocyte(CTL)in peripheral blood and liver tissues of patients with chronic hepititis B(CHB)and its clincial significance.Methods One hundred and fifty-seven HBV-infected patients,including 20 cases of acute hepatitis B,115 cases of chronic hepatitis B,and 22cases of HBV-related liver cirrhosis,and 20 healthy controls were enrolled in this study.Peripheral blood was collected and liver tissues were obtained from some of the enrolled subjects.The CD4+CD25+regulatory T cells and HBV specific CTL were analyzed using flow cytometry and cytokine flow cytometry(CFC).The comparison between groups was done by t test.Results The percentages of CD4+CD25+ regulatory T cells in the peripheral blood of patients with acute hepatitis B and CHB of mild,moderate and severe degree were(2.87±0.94)%,(3.53±1.56)%,(4.59±2.98)%and(3.65±1.73)%,respectively,which were higher than that of controls(2.36±0.60)%(t=2.04,5.97,3.30 and 3.17,respectively,P<0.01).The percentages of HBV specific CTL in the peripheral blood of patients with mild,moderate and severe degree of CHB and HBV-related liver cirrhosis were (0.189±0.152)%,(0.103±0.110)%,(0.118±0.120)%and(0.098±0.101)%,respectively,which were significantly lower than that of acute hepatitis patients [(0.815±0.360)%](t=10.09,11.87,9.17 and 8.96,respectively,P<0.01).CD4+CD25+ regulatory T cells and HBV specific CTL in liver tissues were both higher than those in the peripheral blood.Conclusion CD4+CD25+regulatory T cells may play an important role in anti-HBV immune response through inhibiting CD8+T eell function.

Objective To study the spatial variation of T2 relaxation time of cartilage of knee in healthy adults.Methods T2 values of cartilage of knee in 21 asymptomatic young male adults ( age ranged 24 to 39 years ; mean age , 30 years) were calculated by using a multiecho,spin-echo MR imaging sequence at 1.5T MR scanner on sagittal T2 maps,including the patellar,distal femoral weight and non-weight-bearing as well as proximal tibial weight-bearing cartilages,the differences in the spatial variation between them were analysed using F test.Results All 21 asymptomatic volunteers demonstrated a consistent pattern of spatial variation of T2 values cartilage of knee with longer T2 values near the subchondral bone,decreased in deep zoon and increased in articular surface , there was difference between them (F=70.892 , P＜0.05 ) . The greates spatial variation occurred in the patella.T2 value(26.56 ms±4.4 ms) in the deeper zoon of cartilage of the patella was obviously lower than that of the weight-and non-weight-bearing articular cartilage (P = 0.001 ) . Lateral femoral weight-bearing articular cartilage showed lower T2 value ( 35.2 ms ± 6.31 ms) in the outer transitional superficial zone than thatof the patella and non-weight-bearing cartilage,P=0.002,P=0.000 respectively.Lateral tibial weight-bearing articular cartilage showed showed lower T2 value(37.11 ms±6.6 ms)in the outer transitional superficial zone than that of non-weight-bearing cartilage(P=0.000). Conclusion The spatial variation of T2 relaxation time of cartilage of knee in the vivo in the asymptomatic young adults is like slightly concave curve at 1.5T MR system,that is of reference value in study of degenerative osteoarthritis.

Objective To explore the relationship between the red cell volume distribution width (RDW)in patients with ges-tational diabetes and insulin resistance(IR).Methods A total number of 160 pregnancies performed in Guangzhou Women and Children’s Medical Center from January to October 2016.Matching 80 patients with gestational diabetes (the GDM Group)with 80 healthy pregnancies according to their age,gestational weeks and times.Their inflammatory parameters (hs-CRP,neutrophil-to-lymphocyte ratio,RDW)were respectively examined.HOMA-IR was calculated by testing the level of FBG and FIns.The association of the red cell volume distribution width with insulin resistance was analyzed.Results The level of four inflammatory parameters (hs-CRP,WBC,neutrophil-to-lymphocyte ratio,RDW)were significantly higher than that the control group (t=5.695,5.232,3.337,7.814,all P<0.01).Pearson correlated analysis suggested that RDW had positive correlation with HOMA-IR (r=0.58,P<0.01).Conclusion The RDW level were elevated in the GDM patients and had positive correlation with HOMA-IR,which indicated that IR was associated with inflammation and provided proof to research mechanism of GDM.

BACKGROUND:The main clinical manifestation of senile arteriosclerosis obliterans is lower limb ischemia, which is currently difficult to treat. One method is by autologous stem cell transplantation into the muscles of ischemic limbs to improve the formation of new capillaries and restore lower limb blood flow. Endothelial progenitor cell marker CD34+ cell transplantation has been shown to promote angiogenesis in ischemic limbs. Therefore, we propose that peripheral blood autologous CD34+ cell transplantation in older adult patients with atherosclerotic ischemia could effectively promote angiogenesis.OBJECTIVE:To assume that peripheral blood autologous CD34+ cell transplantation in the elderly with atherosclerotic ischemia could effectively promote angiogenesis.METHODS:This is a prospective, single-center, open-label, randomized, and controlled clinical trial that will be completed at the Qingdao No. 9 People's Hospital, China. Twenty older adult patients with atherosclerotic lower limb ischemia will be randomized into two groups. In the cell transplantation group (n=10), peripheral blood CD34+ cells transfected with vascular endothelial growth factor 165 (VEGF165) gene will be intramuscularly transplanted into the ischemic limbs in older adult patients with atherosclerotic lower limb ischemia. In the control group (n=10), normal saline will be intramuscularly injected into the ischemic limbs. All patients will be followed up for 6 months. The primary outcome will be ankle-brachial indices before and 6 months after transplantation to assess lower limb ischemia in both groups.The secondary outcomes will be the number of microvessels in the lower limb muscles before and 6 months after transplantation, the morphology of new blood vessels revealed by CT angiography, the number of VEGF-immunoreactive cells 6 months after transplantation and the incidence of adverse reactions. The trial was registered at the ClinicalTrials.gov (identifier:NCT03098771), and the study protocol was approved by the Ethics Committee of Qingdao No. 9 People's Hospital of China. All protocols will be in accordance with Declaration of Helsinki,formulated by the World Medical Association. All patients will be informed of study protocols and provide a written informed consent prior to the beginning of the trial.DISCUSSION:This trial will begin in January 2018 and finish in December 2019. We aim to quantify the effects of VEGF165 gene-modified CD34+ cell transplantation in the treatment of older adult patients with atherosclerotic ischemia to develop a new effective treatment of lower limb ischemia.