1.Nucleic Acid Changes in Hemopoietic Tissues of Rats after Combined Radiation-Burn Injury
Journal of Third Military Medical University 1983;0(04):-
Rats were subjected to 15% body surface area third degree burns combined with 2 , 4 , 6 , and 8 Gy irradiation respectively. 48 hours after injury, the DNA and RNA contents of the bone marrow, lymph nodes, spleen and peripheral blood were found to have marked reduction, which became more marked when the dosage of the radiation was increased. Furthermore, it was found that the DNA content decreased more rapidly than the RNA content and the chnage of RNA was more marked in the hemopoietic tissues than in the peripheral blood. Nucleic acid reduction occurred more severely following 2 Gy irradiation than following 15% BSA third degree burns. After the combined iujury of 8 Gy irradiation and burns, th? reduction of nucleic acids in all the above-mentioned tissues exceeded 50%.The mechanism of the changes of necleic acids in the hemopoietic tissues after combined injury of radiation and burns was discussed. Radiation played a more important role than burns in resulting in the reduction but the synergistic effect of burns could not be neglected and could be clearly demonstrated.
2.Experimental study on VEGF165 transferred dermal multipotential stem cells
Zhijun LIU ; Hui XU ; Yongping SU ; Xinze RAN ; Chuanshan XU
Journal of Third Military Medical University 2003;0(18):-
Objective To obtain skin seed cells which can highly express active vascular endothelial growth factor 165(VEGF165) so as to promote refractory wound healing by gene therapy in combination with cell engineering.Methods After pIRES2-EGFP-hVEGF165 was transfected into dermal mesenchymal stem cells(DMSCs) by lipofectin,the expression of VEGF mRNA was detected by RT-PCR,VEGF protein in the supernatant and in the cells were assayed by enzyme-linked immunosorbent assay(ELISA) and Westen blotting respectively.To evaluate the activity of VEGF secreted by transfected DMSCs,hECV304 was cultured with the supernatant of transfected DMSCs and its proliferation activity was analyzed by MTT assays.Meanwhile,the proliferation activity of VEGF-transfected DMSCs and non transfected DMSCs was investigated by MTT.Results The results of RT-PCR,ELISA and Western blot demonstrated that the expression of VEGF in transfected DMSCs was about 1.6 times than that of control DMSCs.The product not only enhanced the proliferation of hECV304 but also increased the proliferation of transfected DMSCs.Conclusion The plasmid pIRES2-EGFP-hVEGF165 is successfully transfected into DMSCs with the aid of lipotransfection,and hVEGF165-transfected DMSCs might high-efficiently secrete highly active VEGF165.
3.Inductive effect of muscular traumatic fluid on the proliferation and myogenesis of bone marrow mesenchymal stem cells
Jin WANG ; Chengji LUO ; Xinze RAN ; Limin XU ; Jiang FENG
Chinese Journal of Tissue Engineering Research 2005;9(18):270-272
BACKGROUND: Bone marrow mesenchymal stem cells (BMMSCs) can be induced differentiating into osteoblasts and chondroblasts, DNA methylate depressant 5-azacytidine can induce BMMSCs expressing myogenic regulating factors: Myf5 and myopoietin, which involving in the differentiation of BMMSCs into myoblasts.OBJECTIVE: Muscular traumatic fluid containing the highest protein content was screened out and co-cultured with BMMSCs,in order to explore the inductive effect on the proliferation and myogeneis of BMMSCs.DESIGN : Standardized comparative study.SETTING .:At State Key Laboratory of Trauma, Bums and Combined Injury,Institute of Combined Injury, Medical College of Preventive Medicine, Third Military Medical University of Chinese PLA.MATERIALS: Muscular traumatic model was established on 18 Balb/C pure rats for the extraction of muscular traumatic fluid, the inductive effect of the fluid on BMMSC was then compared with 5-azacytidine.METHODS:This study was carried out at State Key Laboratory of Trauma,Burns and Combined Injury, Institute of Combined Injury, College of Preventive Medicine, Third Military Medical University of Chinese PLA from April 2001 and September 2003. Bradford colorimetric was used to detect the protein content in the muscular traumatic fluid, and the fluid with the highest protein was used to co-culture with BMMSC, whose effect on the proliferation of BMMSC was measured with MTT methods at day 0, 3, 6, 9, 12, 15.RT-PCR technique was used to detect the expression of myopoietin at day 6,with its myogenic effect compared with that of 5-azacytidine.fluid on BMMSC.eration of BMMSC: the proloferative activity of BMMSC in traumatic fluid genic effect of traumatic fluid on BMMSC: myopoietin could not be found expressed in traumatic fluid group, but strongly expressed in 5-azacytidine group.CONCLUSION: Muscular traumatic fluid can promote the proliferation of BMMSC, but has no myogenic effect.
4.Protective effect of atorvastatin on radiation-induced endothelial cell injury
Xinze RAN ; Huaien ZHENG ; Fengchao WANG ; Xi RAN ; Aiping WANG ; Jing HAN ; Yanqi ZHANG ; Jun CHEN
Chinese Journal of Radiological Medicine and Protection 2009;29(2):129-132
Objective To explore the protective effect of atorvastatin on irradiated endothelium and the thrombomodulin(TM)expression.Methods Cultured human coronary artery endothelial cells(HCAEC)and human umbilical vein endothelial cells(HUVEC)were treated by atorvastatin at the final concentration of 10 μmol/ml for 10 min,and then irradiated with 2 and 25 Gy.Cell cycles status and TM expression were quantitatively measured by flow cytometry 24 hours after irradiation.Protein C activation in endothelial cells was also assessod.Results After administration with atorvastatin for 24 h,the TM expression increased by 77%,59% and 61% in normal control group,2 Gy group and 25 Gy group,respectively(t=27.395,26.420,58.065;P=0.000).The protein C levels decreased by 23% and 34% compared with the normal group post-irradiation to 2 and 25 Gy,but increased by 79% and 76% compared with the irradiated control group after administration with atorvastatin.The rates of cell apoptosis decreased by 6% and 16% in 2 Gy and 25 Gy groups,respectively after administration with atorvastatin for 24 h(t=4.178,17.863;P=0.000).Conclusions Atorva statin can protect endothelia cell from irradiation-induced apeptosis by increasing TM expression and protein C activation.
5.Construction of eukaryotic expressing vector pEGFP-N1/PDGF-A for transducting Dermis-derived mesenchymal stem cells
Guohe YAN ; Yongping SU ; Junping WANG ; Daijie WANG ; Guoping AI ; Fengchao WANG ; Xinze RAN ; Tianmin CHENG
Journal of Third Military Medical University 2003;0(20):-
Objective To clone platelet-derived growth factor A chain (PDGF-A) gene and insert PDGF-A gene into. Enhanced green fluorescent protein (EGFP) vector and then transformed into dermis-drived mesenchymal stem cells (DMSCs). Methods cDNA clones encoding human PDGF-A gene were isolated from a human hepatoma cell line mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The PCR amplified fragment of PDGF-A gene was cloned into pMD18-T vector. The eukaryotic expression vector pEGFP-N1/PDGF-A was constructed by subcolone PDGF-A gene into pEGFP-N1 vector. PDGF-A gene was transfected into DMSCs with the help of Fugene 6 transfection reagent. Results Full cDNA sequence encoding human PDGF-A gene had been cloned, which sequence was consistent with the reported sequence in GenBank by sequence assaying. Conclusion cDNA sequence encoding human PDGF-A gene was successfully cloned into pEGFP-N1. The transient expression of PDGF-A gene in DMSCs has been realized.
6.Enhancement of distribution of dermal multipotent stem cells to bone marrow in rats of total body irradiation by platelet-derived growth factor-AA treatment
Zhaowen ZONG ; Yongchuan REN ; Yue SHEN ; Yonghua CHEN ; Xinze RAN ; Chunmeng SHI ; Tianmin CHENG
Chinese Journal of Radiological Medicine and Protection 2011;31(4):433-436
Objective To observe whether dermal multipotent stem cells (dMSCs) treated with platelet-derived growth factor-AA ( PDGF-AA )could distribute more frequently to the bone marrow in rats of total body irradiation (TBI).Methods Male dMSCs were isolated and 10 μg/L PDGF-AA was added to the culture medium and further cultured for 2 h.Then the expression of tenascin-C were examined by Western blot, and the migration ability of dMSCs was assessed in transwell chamber.The pre-treated dMSCs were transplanted by tail vein injection into female rats administered with total body irradiation, and 2 weeks after transplantation, real-time PCR was employed to measure the amount of dMSCs in bone marrow.Non-treated dMSCs served as control.Results PDGF-AA treatment increased the expression of tenascin-C in dMSCs, made (1.79 ± 0.13) × 105 cells migrate to the lower chamber under the effect of bone marrow extract, and distributed to bone marrow in TBI rats, significantly more than ( 1.24 ± 0.09) ×105 in non-treated dMSCs (t = 8.833, P < 0.0l ).Conclusions PDGF-AA treatment could enhance the migration ability of dMSCs and increase the amount of dMSCs in bone marrow of TBI rats after transplantation.
7.Expression of recombinant human ?-defensin 2 in dermal multipotent stem cells and its antiseptic activity
Nan LI ; Taoyuan XIAO ; Yongping SU ; Hui XU ; Junping WANG ; Zhaowen ZONG ; Xinze RAN ; Shiwu DONG ; Zhijun LIU
Journal of Third Military Medical University 1983;0(04):-
Objective To examine the expression of human ?-defensin 2 (hBD_ 2 ) recombinant adenovirus expression vector in rat dermal multipotent stem cells (dMSCs) and to observe the antiseptic activity of recombinant hBD_ 2 . Methods The expression of hBD_ 2 in dMSCs was examined by RT-PR, fluorescent immunochemistry and Western blotting, and the concentration of recombinant hBD_ 2 in supernate was measured by ELISA. The antiseptic activity of recombinant hBD_ 2 was assessed by K-B disc agar diffusion test. Results hBD_ 2 could be effectively expressed in dMSCs, and the concentration of recombinant hBD_ 2 in supernate was about 743.6 ng/ml . Recombinant hBD_ 2 in supernate showed antiseptic activity. Conclusion Recombinant adenovirus expression vector of hBD_ 2 could be effectively expressed in dMSCs, and the recombinant hBD_ 2 in supernate showed obvious antiseptic effects toward some standard bacteria lines.
8.Transdifferentiation of allotype BMSCs into hepatocytes in bone marrow chimeric mice
Lianyou WANG ; Hui XU ; Shiwu DONG ; Yongping SU ; Xueli PANG ; Dengqun LIU ; Junping WANG ; Xinze RAN ; Fengchao WANG
Journal of Third Military Medical University 2003;0(11):-
Objective To detect whether mice bone marrow mesenchymal stem cells(BMSCs)can contribute to the regeneration of hepatocytes in bone marrow chimeric mice.Methods Female recipient mice(C57BL/6J)underwent whole body gamma-ray irradiation with a dose of 10 Gy to ablate their bone marrow,followed by immediate tail vein injection of BMSCs isolated from male GFP transgenic mice.Animals were killed at different phase points:1 week,1 month,and 3 months.Using fluorescence microscope we directly observed GFP-positive cells in the liver frozen sections,and we also prepared the parafilm sections to detect the GFP-positive cells and the coexpression of GFP and Alb,CK18 by immunohistochemistry and immunofluorescence respectively.Results We found numerous GFP-positive cells in recipient mice liver at 1 week after BMSCs transplantation,some at 1 month and seldom at 3 months.There were some cells coexpressing GFP and Alb,CK18 at all the phase points.Conclusion Allotype BMSCs can differentiate into Alb and CK18 positive hepatocytes in bone marrow chimeric mice,which will become an ideal cell resource for liver tissue project.
9.Feasibility of MSCs mobilization by G-CSF and its prosthetic effect in traumatic brain injury
Jun DENG ; Guoping AI ; Taoli ZHOU ; Junping WANG ; Hui XU ; Zhongmin ZOU ; Shiwu DONG ; Lei HAO ; Xinze RAN ; Yongping SU
Journal of Third Military Medical University 2002;0(12):-
Objective To explore the feasibility of mobilization circulating MSCs by G-CSF and observe the repairing effect of G-CSF mobilization in severe mouse traumatic brain injury(TBI) model.Methods MSCs-derived bone marrow and peripheral blood(PB) were cultured and its CFU-F were counted after mobilization by G-CSF.At 2,24,48,96,120,144,192,264,336 h after severe TBI in mice was establish,the neurobehavior of mice was measured by neurological examination and motor functional test,and mortality rate and pathologic changes were analyzed.Results MSCs-derived PB were successfully cultured.The CFU-F of mobilization group increased significantly than that of control group(P
10.Human amniotic membrane loaded with porcine keratinocytes for constructing epidermal substitute of skin
Guohe YAN ; Yongping SU ; Feng WANG ; Guoping AI ; Tianmin CHENG ; Huaien ZHENG ; Xinze RAN ; Hong XIAO ; Chongfu TAN
Chinese Journal of Tissue Engineering Research 2005;9(22):245-247
BACKGROUND: As a kind of semitransparent membrane, human amniotic membrance contains many kinds of nutrients, which is a good biological material loaded with keratinocytes.OBJECTIVE: To construct epidermal substitute of the skin from human amniotic membrane loaded with porcine keratinocytes and examine the morphological characteristics of the growth and proliferation of keratinocytes seeded on human amniotic membrane.DESIGN: Single sample study and repetitive measured observation based on the cells.SETTING: Institute of Combined Injuries of Chinese PLA, Academy of Preventive Medicine, Third Military Medical University of Chinese PLA.MATERIALS: The experiment was completed in the State Key Laboratory of Trauma, Burn and Combined Injury and Institute of Combined Injuries of Chinese PLA, Academy of Preventive Medicine, Third Military Medical University of Chinese PLA from January to November 2001. Porcine keratinocytes was collected from Guizhou minipigs aged 3 weeks.METHODS: The primarily cultured keratinocytes of Guizhou minipigs were subcuhured, expanded and bred on the stroma surface of human amniotic membrance at the density of 1.63 × 105/cm2. The growth and proliferation of keratinocytes were observed under inverted microscope every day. From the 3rd day and the 15th day after being cultured, the growth of keratinocytes on human amniotic membrane was examined under light microscope and electron microscope.MAIN OUTCOME MEASURES: The growth of keratinocytes on human amniotic membrane was examined RESULTS: Keratinocytes evidently adhered to the stroma surface of human amniotic membrane about 30 minutes after being cultured, which was observed under inverted microscope. Most keratinocytes grew and adhered to the stroma surface of human amniotic membrane within 24 hours. Monolayer of keratinocytes formed and completely covered human amniotic membrane within 3 days. It was observed under the light microscope that the monolayer of keratinocytes adhered to human amniotic membrane and arrayed tightly. The keratinocytes presented in the shape of polygon, and plasmalemmas of keratinocytes formed many pseudopods under the observation with scanning electron microscope. Keratinocytes adhered to human amniotic membrane well and with many keratinofilaments in them under the observation with transmission electron microscope. Keratinocytes arrayed on human amniotic membrane densely with many cellular debris and some keratinocytes formed cavitations in them due to aging after growth for 15 days under the observation with inverted microscope.CONCLUSION: Human amitotic membrane is a good carrier of keratinocytes cultured on it in vitro, and is able to promote the proliferation of keratinocytes significantly. However, when keratinocytes were loaded on the human amniotic membrane for 15 days, some keratinocytes formed cavitations in them due to aging.