1.Human adipose-derived stem cells promote the survival and function of islet cells in co-culture
Zizhao JIAO ; Wujun XUE ; Xiaohui TIAN ; Yang LI ; Jin ZHENG
Chinese Journal of Organ Transplantation 2015;36(9):549-555
Objective To isolate and culture human adipose-derived stem cells (hADSCs),investigate the influence of hADSCs on the cellular morphology,survival rate,and function of human islet cells under the in vitro non-contact co-culture conditions,and explore its mechanism.Method hADSCs were isolated by collagenase digestion method,then cultured,and identified by morphology,immunofluorescence and multi-directional differentiation.Adult islet cells were separated and purified by Liberase enzyme and Ficoll 400,then divided into co-culture group and individual group.The cellular growth morphology of islet cells was observed by inverted phase contrast microscope.The survival rate of islet cells,insulin secretory volume,insulin stimulation index and concentration of growth factor in the supernatant were compared between the two groups.Result hADSCs of the third generation showed uniform long spindle fibrocyte-like morphology,and had multi-directional differentiational potentials of osteogenesis and adipogenesis.Immunofluorescence test of surface antigens on hADSCs revealed CD44 + and CD49d +,CD31-,CD34-and CD106-.After 14-day culture,the islet cellular morphology in co-culture group was more intact than that in individual group.The survival rate of islet cells in co-culture group was (82.83 + 2.32) %,and that in individual group was (53.00 + 2.82) % (P<0.01).Insulin secretory volumes were (23.66 + 2.11) and (7.82 +1.09) mU/L respectively in co-culture group and individual group under high glucose concentration,and 13.22 + 0.77 and 6.40 + 0.44 mU/L respectively under low glucose concentration (P<0.01 for all).Insulin stimulation index was decreased from 1.67 + 0.10 (at 3rd day) to 1.77 + 0.13 (at 14th day) in co-culture group,and from (1.67 + 0.10) (at 3rd day) to (1.77 + 0.13) (at 14th day) in individual group (P<0.01).After 14-day culture,the concentrations of HGF,TGF-β,VEGF and bFGF in the supernatant were higher in co-culture group than in individual group (P<0.01).Conclusion hADSCs were isolated and cultured successfully from adult adipose tissue.They could increase the survival rate and improve the function of islet cells when co-culture with the adult islet cells in vitro through secreting HGF,TGF-β,VEGF and b-FGF.
2.ADSCs promotes the proliferation and vascularization of HUVECs when co-cultured in vitro
Zizhao JIAO ; Wujun XUE ; Xiaohui TIAN ; Yang LI ; Jin ZHENG
Journal of Xi'an Jiaotong University(Medical Sciences) 2016;37(4):493-499
ABSTRACT:Objective For preparation of vascularized islets , to isolate and culture human adipose derived stem cells , investigate the role of adipose derived stem cells (ADSCs ) in promoting the proliferation and vascularization of human umbilical vein endothelial cells (HUVECs ) co‐cultured in vitro , and explore its mechanism .Methods ADSCs and HUVECs were isolated by collagenase digestion method ,then cultured ,and identified by morphology ,immunofluorescence or multi‐directional differentiation .The co‐culture system of ADSCs and HUVECs was established , HUVECs cultured alone were set up for control group . The proliferation , vascularization and concentration of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b‐FGF)in the supernatant were compared between the two groups .Results The third generational ADSCs had uniform long spindle fiberous morphology and multi‐directional differentiational function . Immunofluorescence test of surface antigens on ADSCs revealed CD44/CD49d (+ ) ,CD31/CD34 (-) ,on HUVECs CD31/vWF (+ ) . High vascular density was found when co‐cultured in Matrigel of ADSCs and HUVECs than alone of HUVECs .Growth curve shown at days 3 , 4 and 5 of the logarithmic phase , HUVECs count in co‐culture group of ADSCs and HUVECs was (4 .52 ± 0 .31) × 104 ,(7 .18 ± 0 .45) × 104 ,and (8 .23 ± 0 .36) × 104 under indirect co‐culture condition , while that in individual HUVECs group was (2 .71 ± 0 .25) × 104 ,(4 .87 ± 0 .26) × 104 ,and (6 .86 ± 0 .33) × 104 ( P<0 .01) .Population doubling time of HUVECs was shorter in co‐culture group than in individual group .Also ,the OD value of HUVECs was higher in co‐culture group than in individual group when cultured at days 1 ,3 ,5 and 7 ( P<0 .01) .When cultured at days 3 ,7 and 13 ,the concentration of VEGF and b‐FGF in the supernatant was higher in co‐culture group than in individual group ( P< 0 .01 ) . Conclusion ADSCs can promote the proliferation and vascularization of HUVECs in vitro co‐culture conditions by secreting or increasing the HUVECs secretion of VEGF and b‐FGF .
3.Clinical Cross Sectional Study of Herpes Simplex Virus Infection of Central Nervous System in Newborn Infants
tian-jiao, YANG ; qi-rong, ZHU ; xiao-hong, WANG ; jian-she, WANG
Journal of Applied Clinical Pediatrics 2004;0(12):-
Objective To investigate the prevalence of herpes simplex virus(HSV) infection of central nervous system(CNS) in newborn infant,and analyze its clinical characteristics.Methods Cerebrospinal fluid(CSF) was collected from 40 acute viral infection of central nervous system who were hospitalized during June 2001 to June 2002.Polymerse chain reacton techniques(nested-PCR)was used to detect HSVspecific DNA in CSF,enzyme-linked immunosorbert assays(ELISA)was applied to detect HSV-specific IgM and IgG antibody in CSF and serum specimens.Results Two cases of neonatal patients were HSV-1 DNA PCR positive in CSF,both(mo)-ther were normal during pregnancy without a history of genital herpes.Clinical presentations of one case belonged to disseminated HSV infections and the other was limited to CNS infections.HSV-2 DNA PCR of 40 cases of neonatal patients were negative in CSF.Conclusions The rate of HSV neonatal CNS infection was 5% among viral neonatal CNS infections.HSV type 1 in the period,which showed that HSV type 1 may be the common type of HSV neonatal CNS infections.The result seems to be related to low prevalence for HSV-2(among) pregnancy women in China.
4.THE EFFECT OF rAAV-hGDNF ON PROTECTING RAT SPINAL CORD NEURONS FROM DEATH INDUCED BY GLUTAMATE
Jianwei JIAO ; Yang GAO ; Jingsheng TIAN ; Xiaobing WU ; Zhijian WU ; Jiayo LIN
Acta Anatomica Sinica 2002;0(05):-
Objective In this experiment,we explored the effects of rAAV\|hGDNF on protecting spinal neurons From death. Methods rAAV\|hGDNF particals were produced by recombinant virus technolgy,and infected the culture spinal neurons which were exposed to glutamate.We counted the mortality rate and detected the expression of NOS mRNA by RT\|PCR. Results In the group transfering rAAV\|hGDNF the death rate was inhibited(50%?0\^02,control 59\^25%?0\^023, P
5.Research progress on triterpenoids of Betula plants
Yan-xin LI ; Ting GONG ; Jing-jing CHEN ; Tian-jiao CHEN ; Jin-ling YANG ; Ping ZHU
Acta Pharmaceutica Sinica 2023;57(5):1211-1220
The secondary metabolites of plants are important sources of natural drugs.
6.Study on Context of Chinese Medicine School Education in Modern Zhejiang Province
Journal of Zhejiang Chinese Medical University 2018;42(9):752-755
[Objective] To discuss and analyze the development history of TCM education in Zhejiang in modern times and provide valuable experience for modern TCM education theory and practice. [Methods] The article goes through the historical development of TCM education in modern Zhejiang, the general situation of TCM education, and the characteristics of TCM education. It deepens historical materials to search, collate and excavate the precious literature resources of historical materials and summarizes them. [Results] The modern TCM, Beiyang government, Nanjing National Government and other periods of traditional Chinese medicine business were in a difficult stage, but TCM education in these periods was actively going forward in the reform. The modern Chinese medicine school runs a comprehensive reform from the aspects of the school-running concept, enrollment system, school system and curriculum setting to traditional TCM education, creating a unique school-running model and promoting the development of TCM education. [Conclusion] Modern Chinese medicine education in Zhejiang has developed in twists and turns. Its experience has provided valuable experience for the theory and practice of Chinese medicine education the present age. promoting the development of TCM education. [Conclusion] Modern Chinese medicine education in Zhejiang has developed in twists and turns. Its experience has provided valuable experience for the theory and practice of Chinese medicine education the present age.
7.Identification and transcriptional activity analysis of core regulatory region of human guanylate binding protein 5 gene promoter
YE Ting ; YANG Kang ; WANG Tian-tian ; LIAO Yu-jiao ; DU Wen-qian ; HUANG Min ; JIANG Pei-wen ; LI Min-hui ; YANG Ping
Chinese Journal of Biologicals 2023;36(2):138-144
Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1 623 ~ +47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1 623 ~ +47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520 ~ +47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1 623 ~-520 bp of 5 'UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1 623 ~-520 bp of the 5 'UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GBP5 promoter.
8.Apolipoprotein A-I mimetic peptide D4F protects macrophages from oxi-dized low-density lipoprotein-induced apoptosis by inhibiting caspase-12
Hua TIAN ; Yanyan LI ; Mingde DING ; Nana YANG ; Peng JIAO ; Hui SANG ; Yongqi FANG ; Shutong YAO ; Shucun QIN
Chinese Journal of Pathophysiology 2015;(10):1750-1755
[ABSTRACT]AIM:ToinvestigatetheeffectofD4F,anapolipoproteinA-Imimeticpeptide,onoxidizedlow-density lipoprotein ( ox-LDL)-induced macrophage apoptosis and activation of caspase-12, a key molecule in endoplasmic reticulum stress ( ERS )-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms. METHODS:RAW264.7 macrophages were pretreated with D4F (12.5, 25 and 50 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium ( DPI, 5 μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin ( TM, 4 mg/L) for 24 h.The cell viability and apoptosis were determined by MTT assay and TUNEL detection, respective-ly.The levels of malondialdehyde ( MDA) and reactive oxygen species ( ROS) in the cells and the activities of superoxide dismutase ( SOD) and nicotinamide adenine dinucleotide phosphate ( NADPH) oxidase were determined.The protein level of caspase-12 was examined by Western blot analysis.RESULTS: Similar to the ERS inhibitor PBA, D4F protected RAW264.7 macrophages from ox-LDL or TM ( an ERS inducer)-induced decrease in the viability and increase in apoptotic rate in a dose-dependent manner.Like DPI (an oxidative stress inhibitor), D4F significantly inhibited ox-LDL-induced ox-idative stress, as expressed by the decreased generation of ROS and MDA ( P<0.01) , the increased activity of SOD and the decreased activity of NADPH oxidase (P<0.05).Moreover, similar to PBA and DPI, D4F significantly suppressed ox-LDL-induced activation of caspase-12 in a concentration-dependent manner ( P<0.05) .Furthermore, D4F also inhibi-ted the caspase-12 activation induced by TM (P<0.05).CONCLUSION: D4F inhibits macrophage apoptosis induced by ox-LDL, and the mechanism is at least partially by reducing oxidative stress and inhibiting the activation of caspase-12.
9.Immunomodulatory effect of Wharton's jelly-derived mesenchymal stem cells from human umbilical cord on human peripheral blood T lymphocytes
Changhui ZHOU ; Yi TIAN ; Bo YANG ; Xiang HU ; Hongliang JIAO ; Yunfan ZHOU ; Chengchun WANG ; Chenxi GU ; Ningjing LEI ; Fangxia GUAN
Chinese Journal of Tissue Engineering Research 2010;14(14):2485-2491
BACKGROUND:Bone marrow mesenchymal stem cells have low immunogenicity and immunomodulatory effect,but there are seldom reports concerning the immunomodulatory effect of Wharton's jelly-derived mesenchymal stem cells of human umbilical cord and its mechanims.OBJECTIVE:To investigate the immunomodulatory effects and mechanisms of Wharton's jelly-derived mesenchymal stem cells of human umbilical cord on varient peripheral blood T lymphocytes.METHODS:Mesenchymal stem cells were isolateded from Wharton's jelly of human umbilical cord by tissue culture.T lymphocytes from human peripheral blood were stimulated by phytohemagglutinin and co-cultured with umbilical cord Wharton's jelly-derived mesenchymal stem cells and umbilical cord Wharton's jelly-derived mesenchymal stem cells supernatant respectively to measure A value following 72 hours of coculture using multifunctional microplate reader.Expression of cytokines including transforming growth factor-beta 1(TGF-β1)and interferon-y(IFN-γ)was evaluated by enzyme-labeled immunosorbent assay.RESULTS AND CONCLUSION:Wharton's jelly-derived mesenchymal stem cells could inhibite the proliferation of T lymphocytes induced by phytohemagglutinin.The proliferation inhibition rate was 56%(P<0.01).Wharton's jelly-derived mesenchymal stem cells supernatant also had inhibitory effects on proliferation of T lymphocytes induced by phytohemagglutinin,in a dose-dependent fashion.The proliferation inhibition rates were 8.3% and 27% respectively in the 50% Wharton's jelly-derived mesenchymal stem cells supernatant and 100% Wharton's jelly-derived mesenchymal stem cells supematant groups(P<0.05).Wharton's jelly-derived mesenchymal stem cells significantly decreased γ-interferon secrted from T-lymphocytes(P<0.05).The secretion of TGF-β1 was lower in the coculture of Wharton's jelly-derived mesenchymal stem cells and T lymphocytes group than Wharton's jelly-derived mesenchymal stem cells alone group(P<0.05).These indicated that Wharton's jelly-derived mesenchymal stem cells and Wharton's jelly-derived mesenchymal stem cells supernatant have inhibitory effects on proliferation of T lymphocytes induced by phytohemagglutinin.The mechanims may be associated with cell contant and inhibition of v-interferon secrted from T-lymphocytes.
10.Effect of bone marrow mesenchymal stem cells transfected by Zrt/Irt-like protein 1 on adipogenic differentiation via tumor necrosis factor alpha signaling pathway
Xiaoyun ZHANG ; Jinhuan LI ; Zhenzhong YUAN ; Yueping CHEN ; Tian XIA ; Yinghong ZHUO ; Yang FENG ; Jiao LAN ; Panfeng DONG ; Bin ZHAO
Chinese Journal of Tissue Engineering Research 2017;21(13):1986-1991
BACKGROUND:Previous studies have confirmed that ethanol can promote adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and up-regulate the expression of PPARγ and aP2 in the tumor necrosis factor α (TNF-α) signaling pathway. As a member of the ZIP protein family, Zrt/Irt-like protein 1 (ZIP1) is closely related to bone metabolism and osteogenic differentiation.OBJECTIVE:To study the effect of BMSCs transfected by ZIP1 on TNF-α signaling pathway in the process of adipogenic differentiation.METHODS:The BMSCs from rabbits were isolated and cultured under different concentrations of alcohol (0.03, 0.09,0.15, 0.21 mol/L), followed by transfection by ZIP1 siRNA and ZIP1 expression vector.RESULTS AND CONCLUSION:After culture in alcohol, the expression levels of aP2 and PPARγ proteins were both significantly increased (P < 0.05), and the level of triglyceride was increased in all alcohol groups except for 0.03 mol/L alcohol group (P < 0.05). After siRNA transfection, the expression levels of aP2 and PPARγ as well as the level of triglyceride were increased significantly in all the alcohol groups (P < 0.05); however, ZIP1 transfection decreased the expression levels of aP2 and PPARγ proteins (P < 0.05). To conclude, ZIP1 siRNA could promote the adipogenic differentiation of BMSCs through the activation of TNF-α signaling pathway.