Therapeutic effect of regulatory T cells on mice with experimental autoimmune encephalomyelitis / 中华微生物学和免疫学杂志

Objective@#To study the role of Treg cells in the development of mouse experimental autoimmune encephalomyelitis (EAE) through depleting or transplanting Treg cells.@*Methods@#C57BL/6 mice were injected with anti-CD25 monoclonal antibody to deplete natural CD25-expressing Treg cells in vivo, and then treated with MOG35-55/CFA to induce EAE. Their EAE scores were compared with those of the mice without Treg cell deletion (control group). The numbers and percentages of CD4+ Foxp3+ cells in mouse blood samples on 6 d, 10 d, 20 d and 35 d were quantified using flow cytometry. To evaluate the therapeutic effect of Treg cells transplantation on EAE, magnetic activated cell sorting (MACS) combined with fluorescence-activated cell sorting (FACS) was used to isolate Treg cells from spleen and lymph nodes of Foxp3GFP+ transgenic mice on 6 d after EAE induction. Then the cells were injected through tail vein into wild-type mice on 6 d after EAE induction. The EAE scores of both recipient and control mice were recorded and compared.@*Results@#The efficiency of natural Treg cells depletion with anti-CD25 antibody was above 95%. The mice with Treg cell depletion developed significantly more severe EAE than the control mice after MOG35-55/CFA induction. FACS analysis of Treg cells during the development of EAE demonstrated that the lowest Treg cell percentage was detected on 6 d after EAE induction, hence it was the time point for the transplantation of Treg cells. CD4+ GFP+ Treg cells were isolated from Foxp3GFP+ transgenic mice on 6 d after EAE induction and immediately transplanted into wild-type mice on 6 d after EAE induction. The transplantation of isolated Treg cells significantly alleviated the EAE in mice as compared with the control group.@*Conclusions@#Mice with Treg cell depletion developed severer EAE than the control mice after induction, but the EAE score could be significantly reduced with the transplantation of Treg cells. This study showed that the transplanted Treg cells had protective effect on mice during the course of EAE development. Thus, Treg cell transplantation could be used as an effective therapeutic approach for the treatment of multiple sclerosis (MS).

Therapeutic effect of regulatory T cells on mice with experimental autoimmune encephalomyelitis / 中华微生物学和免疫学杂志

Objective To study the role of Treg cells in the development of mouse experimental autoimmune encephalomyelitis (EAE) through depleting or transplanting Treg cells. Methods C57BL/ 6 mice were injected with anti-CD25 monoclonal antibody to deplete natural CD25-expressing Treg cells in vi-vo, and then treated with MOG35-55 / CFA to induce EAE. Their EAE scores were compared with those of the mice without Treg cell deletion ( control group). The numbers and percentages of CD4+ Foxp3+ cells in mouse blood samples on 6 d, 10 d, 20 d and 35 d were quantified using flow cytometry. To evaluate the therapeutic effect of Treg cells transplantation on EAE, magnetic activated cell sorting (MACS) combined with fluorescence-activated cell sorting (FACS) was used to isolate Treg cells from spleen and lymph nodes of Foxp3GFP+ transgenic mice on 6 d after EAE induction. Then the cells were injected through tail vein into wild-type mice on 6 d after EAE induction. The EAE scores of both recipient and control mice were recorded and compared. Results The efficiency of natural Treg cells depletion with anti-CD25 antibody was above 95％ . The mice with Treg cell depletion developed significantly more severe EAE than the control mice after MOG35-55 / CFA induction. FACS analysis of Treg cells during the development of EAE demonstrated that the lowest Treg cell percentage was detected on 6 d after EAE induction, hence it was the time point for the transplantation of Treg cells. CD4+GFP+ Treg cells were isolated from Foxp3GFP+ transgenic mice on 6 d after EAE induction and immediately transplanted into wild-type mice on 6 d after EAE induction. The transplan-tation of isolated Treg cells significantly alleviated the EAE in mice as compared with the control group.Conclusions Mice with Treg cell depletion developed severer EAE than the control mice after induction, but the EAE score could be significantly reduced with the transplantation of Treg cells. This study showed that the transplanted Treg cells had protective effect on mice during the course of EAE development. Thus, Treg cell transplantation could be used as an effective therapeutic approach for the treatment of multiple scle-rosis (MS).

Progress of induced pluripotent stem cell technology in the research of Parkinson′s disease / 医学研究生学报

[Abstract ] In recent years, induced pluripotent stem cells (iPSCs) technology has played an important role in basic and clini-cal application research of Parkinson′s disease ( PD) and acquired significant progress .The neural progenitor /stem cells or dopamine ( DA) neurons which were obtained through iPSCs technique and direct differentiation technique from somatic cells were used for the study of cell therapy in PD , and good results were achieved .The cell models of DA neurons were established from PD patients carrying LRRK2, PAKK2, PINK or SNCA mutations via iPSCs technology , and the mitochondrial function and morphology , oxidative stress,α-synuclein ( SNCA) accumulation , and other aspects were studied on the pathogenesis of PD .This article briefly reviews the latest pro-gress of iPSCs technology in transplantation for treatment of PD and the establishment of cell model of PD disease , and provides refer-ence for further research .

Cellular uptake of Tumor-targeted nanoparticles derived from pullulan acetate conjugate / 医学研究生学报

Objective Folate receptors ( FRs) , overexpressed on the surface of a variety of tumor cells , are potential targets for tumor targeting therapy .This study aimed to prepare an FR-mediated drug nanocarrier with folate conjugated pullulan acetate ( FPA) to target chemotherapeutic agents to FR-overexpressed tumor cells and investigate its tumor-suppressing effect in vitro. Methods The cytotoxicity of epirubicin-loaded FPA nanoparticles ( FPA/EPI NP) against HepG2 and Hela cells was evaluated by MTS assay.The HepG2 and Hela cells were divided into five groups to be treated with NPs (NP control), chlorpromazine, chloro-quine, amiloride, and folate, respectively, followed by detection of the fluorescence intensity by flow cytometry . Results FPA/EPI NP was successfully formulated into NPs , with the mean particle size of (268.5 ±12.0) nm, by dialysis with an almost spherical shape . The drug-loading rate and entrapment rate of FPA/EPI NP were (6.45 ±1.04) and (72.45 ±11.50) %, respectively.The survival rates of the HepG2 and Hela cells were both >95%after 24 hours of incubation with FPA NP at 5, 40, 200, 400 and 1000μg/mL and 90.0%after 72 hours.The survival rates of the HepG2 cells treated with 5, 40, 200, 400 and 1000μg/mL FPA/EPI NP for 24 hours were (92.3 ±5.2), (70.4 ±4.6), (50.0 ±4.0), (41.1 ±4.1) and (27.0 ±3.6) %, respectively.Compared with the NP control group, the Hela cells of the chlorpromazine , amiloride and folate groups showed a significantly lower rate of NP uptake (P<0.05), and so did the HepG2 cells pretreated with chlorpromazine or amilo-ride (P<0.05).At 72 hours, the half maximal inhibitory concentrations (IC50) of FPA/EPI NP against HepG2 and Hela cells were 168 and 105μg/mL, respectively . Conclusion Clathrin-mediated endocytosis and macropinocytosis are involved in the internaliza-tion of FPA/EPI NP in HepG2 cells, while clathrin-and FR-mediated endocytosis in that of Hela cells .FPA NP may serve as a new drug carrier for tumor-targeted therapy .

Construction and identification of pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector / 中国组织工程研究

BACKGROUND:Human glial cellline-derived neurotrophic factor (GDNF) and vascular endothelial growth factor 165 (VEGF 165 ) are essential genes for celldifferentiation. OBJECTIVE:To construct and identify pIRES 2-GDNF-VEGF 165 bicistronic eukaryotic expression vector. METHODS:Human GDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then the GDNF cDNA fragment was inserted into the multiple cloning sites of pIRES 2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES 2-GDNF-EGFP. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by twin PCR. Then VEGF 165 cDNA fragment was cloned into the pIRES 2-GDNF-EGFP, instead of EGFP, to create a double gene co-expressing vector plasmid pIRES 2-GDNF-VEGF 165 containing internal ribosome entry sites. Then pIRES 2-GDNF-VEGF 165 was used to transfect HEK293 cells. RT-PCR and western blot analysis were performed to test the co-expression of double genes. RESULTS AND CONCLUSION:DNA sequencing analysis demonstrated that the GDNF and VEGF 165 were exactly consistent with the sequence recorded in the GenBank. The size of GDNF gene was 636 bp and the size of VEGF165 gene was 576 bp. Enzyme digestion analysis indicated that, pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector inserted GDNF band by Bgl II/Bam HI, inserted IRES-VEGF 165 fragment by Bam HI/Not I, and inserted GDNF-IRES-VEGF165 fragment by Bgl II/Not I. RT-PCR and western blot analysis showed that, after HEK293 cells were transfected with pIRES 2-GDNF-VEGF 165 , double genes were expressed at the mRNA and protein levels. The pIRES 2-GDNF-VEGF 165 bicistronic eukaryotic expression vector is successful y constructed.

Construction and Identification of pIRES 2-BDNF-VEGF 165 bicistronic eukaryotic expression vector / 中国组织工程研究

BACKGROUND:Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor 165 (VEGF 165 ) are essential genes for celldifferentiation. Virus mediated method has been used numerously in researches, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question. OBJECTIVE:To construct and identify pIRES 2-BDNF-VEGF 165 bicistronic eukaryotic expression vector. METHODS:BDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then, the BDNF cDNA fragment was inserted into the multiple cloning sites of pIRES 2-EGFP to generate the bicistronic eukaryotic expression plasmid pIRES 2-BDNF-EGFP. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by double PCR. Next step was that VEGF 165 cDNA fragment was cloned into the pIRES2BDNF-EGFP instead of EGFP to create a double gene co-expressing vector plasmid pIRES 2-BDNF-VEGF 165 . Then, pIRES 2-BDNF-VEGF 165 was used to transfect HEK293 cells, and RT-PCR and western-blot assay were employed to test the co-expression of double genes. RESULTS AND CONCLUSION:BDNF and VEGF 165 genes were cloned in this study. The DNA sequencing analysis demonstrated that the BDNF and VEGF 165 were exactly consistent with the sequence recorded in the GenBank. The size of BDNF gene was 744 bp. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by PCR, and the size of VEGF 165 gene was 576 bp. Enzyme digestion analysis indicated that BDNF and VEGF 165 genes were inserted into the expression vector pIRES 2-EGFP correctly and the BDNF and VEGF165 co-expression plasmid was successful y constructed. Then, by transfecting pIRES 2-BDNF-VEGF 165 into HEK293 cells, double genes were expressed at the mRNA and protein level. It provides a novel expression system, which enables further study on the functions of BDNF and VEGF 165 genes.

Abnormal neural behaviors of neonatal rat due to hyperthermia and low dosage of cigarette exposure during pregnancy / 中国组织工程研究

BACKGROUND: Neural behavior is a sensitive parameter for predicting abnormal central nervous growth that is resulted from multiple factors. Therefore this study was designed to explore the influence of hypothermia and cigarette smoke exposure during pregnancy on neural behavioral growth of neonatal rats.OBJECTIVE: To study the influence of hypothermia and cigarette smoke exposure on neural behavioral growth of neonatal rats.DESIGN: Randomized case control study based on experimental animals. SETTING: Obstetric and Gynecological Department , Third Affiliated Hospital of Xinxiang Medical College; Cytobiological Department of Xinxiang Medical College.MATERIALS: Totally 270 healthy unpregnant SD rats were obtained, including 180 males with body mass of 190- 210 g, and 90 female rats with body mass of 225-275 g. Rats were raised with granulated feed and drinking water routinely in room of 25 ℃ with natural sunlight. Male and female rats in estrus were put in the same cage overnight in proportion of 2: 1, pregnancy was confirmed if vaginal bolt was observed the following morning. Totally 160 pregnant rats were obtained and randomly divided into 16 groups that were exposed to hyperthermia, cigarette smoke or their combination and by this way, earlier reflex, learning and memory of neonatal rats were observed after natural delivery,INTERVENTIONS: From the second day of pregnancy, rats in 37 ℃, 41 ℃,42 ℃ hyperthermia combining cigarette group received subcutaneous injection of 1 mL smoke hydrotrope every other day, while rats in 25 ℃ control group and 37 ℃, 41 ℃, 42 ℃ single hyperthemia groupwere given 2.0 mL double distilled water every day for totally 7 times. From the pregnancy of 10 days, pregnant rats were put into incubator, and rats anus temperature should be kept respectively at(37 ±0.5) ℃, (41 ±0.5)℃, (42 ±0.5)℃in the 37 ℃, 41 ℃, 42 ℃ of single hyperthemia groups and the temperature should last for 2.0 minutes; nats of 37 ℃, 41 ℃, 42 ℃hyperthemia combining cigarette group received the same treatment and were divided into four subgroups with the duration of 0. 5, 1.0, 1.5, 2.0 minutes respectively;rats in 25℃ control group were not exposed to hyperthermia. Rats were then singly raised from the pregnancy of 18 days for natural delivery. Pregnant days, number of neonatal rat and death rats were recorded, and visible malformation was observed. The average body masses of each cote of neonatal rats were measured every week. Time of eye-opening, ear-opening, hair-growing and teething were observed for assessing their physical growth. Reflex and sensory function of neonatal rats, such as planar tuning, spatial turning, visual orientation and auditory surprise was observed at 3, 7, 12, 16 days after birth respectively; The mean targeting time of each cote was used as reference for assessing their feflex. Y-lybirinth test was used for assessing learning and memory function of neonatal rats(30 days after birth).MAIN OUTCOME MEASURES: ① Main outcomes: Reflex and sensory, such as planar tuning, spatial turning, visual orientation and auditory surrise. ② Secondary outcomes: Influence of hyperthemia and cigarette exposure on learning memory function of neonatal rats.RESULTS: For hyperthermia group(41C and 42C), targeting time of neonatal rat physical growth(eye-opening, ear-opening, hair-growing and teething) and reflex sensory function(planar tuning, spatial turning, nvisual orientation and auditory surprise), as well as learning and memory(minute) were obviously delayed in contrast with those of the control group and 37 C group(χ2 = 11.34, P ＜0.01 ); meanwhile, longer hyperthermia exposure (1.5 minutes) combined with cigarette smoke had supplementary effect on the above neural behaviors, which was significantly different from that of single hyperthermia group(χ2=10.29, P＜0.01).CONCLUSION: Abnormal neural behavior of neonatal rats, such as growing retardation, learning and memory obstacle could be induced by the combination of longer hyperthemia exposure and cigarette smoke.