By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 micromol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.
Cartilage/*cytology ; Cell Differentiation/*drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Oligodeoxyribonucleotides/genetics ; Oligodeoxyribonucleotides/*pharmacology ; Rats, Sprague-Dawley ; Stathmin/*genetics ; Stathmin/pharmacology ; Stem Cells/*cytology

Objective:To establish and identify humanized-SCID mouse model(hu-SCID).Methods:SCID mouse was treated by CTX to inhibit the hemocytopoiesis. With successive 4-day injection, human peripheral blood mononuclear cells(PBMC) were engrafted into SCID mouse through intraperitoneal injection. After 4, 8 and 12 weeks of engraftment, peripheral blood, spleen and liver tissues of engrafted SCID mouse were harvested. Human CD3~+, CD19~+ cells in peripheral blood were analyzed by inflorescence microscopy and FCM, human CD3~+, CD19~+ cells in spleen and liver tissues were observed by immune histochemistry, and human IgG level in SCID mouse serum was measured by ELISA.Results:After engraftment of 4, 8 and 12 weeks, human CD3~+, CD19~+ cells in SCID peripheral blood were identified by inflorescence microscopy and the percents were 31% and 10% respectively by FCM analysis. And these cells could be evidenced after 12 weeks later. Through immune histochemistry human CD3~+、CD19~+ cells were detected in mouse spleen but not in liver tissue. Furthermore the titer of human IgG in mouse serum was 390,1 100 and 1 040 ?g/ml at each time point respectively.Conclusion:Our experimental results demonstrated that a bona fide humanized SCID model was established.

Objective:To explore a new method of the cultivation of adoptive immunotherapy cells.Methods: Mononuclear cells was isoplated by density gradient centrifugation and then proliferated by using CD 40-agonist monoclonal antibody 5C11、cytokine of IFN-αand IL-7(CD40 group)in vitro.During the culturing procedure ,the cell morphology was obersved by optical microscope.The percentage of T-lymphocytes, NK-T cells, Treg cells and the cell proliferation, which were compared with CIK group CD3mAb activated,was detected on the 9th day.Results:There was no significant difference of CD 4+/CD8+T cells percentage between the two groups.But the Treg cells percentage of CIK group was far higher than that of CD 40 group,while the percentage of CD3+CD56+NK-T cells was lower than that of CD 40 group.And a group of Mo-NK-DC cells were observed in the CD 40 group.Conclusion: The new method of adoptive immunity therapy has been established in this study could increase the percentage of NK -T cells which had the ability to kill tumor cells.Simultaneously ,it is reduced the amount of Treg cells significantly.

AIM: To prepare and characterize a novel anti-human CD83 monoclonal mAb. METHODS: Female BALB/c mice of 6-8 weeks old were immunized with CD83 transfectant (L929/CD83) as immunogen. The spleen B cells of the mice were fused with Sp2/0 myeloma cells. The hybridoma cells were screened with CD83 transfectant (L929/CD83 and 293/CD83) by FCM. The biological characteristics of antibody were investigated by rapid isotyping analysis, karyotype analysis, competitive inhibition test and Western blot. RESULTS: One hybridoma cell line named 9D8 was obtained, which had the property of secreting antihuman CD83 monoclonal antibody steadily, This mAb specifically recognized CD83 molecule expressed on human mature DC, activated T cells, and tumor cell line Daudi, myeloma cell line 8226. This mAb can recognize a distinct epitope from comercial mAb(HB15e). CONCLUSION: One hybndoma cell line has been developed successfully, which may lay a fundation for further study on the function of this molecule.

V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a member of the B7 family that maintains homeostasis in T cells and myeloid cells.Blocking VISTA inhibits tumor development in in vitro and in vivo trials, and is an important target for tumor immunotherapy.This review focuses on its structural features, expression and biological functions in tumor microenvironment, summarizes the current stage of small molecule inhibitors and antibodies targeting VISTA, and discusses the research approaches.It aims to provide a rationale for subsequent study on VISTA and the development of related immune checkpoint antitumor drugs.

Objective:To investigate the negative effect of the IL-10 on monocyte-derived DC maturation and differation iv vitro,and the potentiation of the TNF-? or sCD40L to inhibit or reverse the IL-10′s inhibitory effect on monocyte-derived DC.Methods:The expression of the surface molecules on DC was detected by FACS analysis.The potentiation to stimulate T cell proliferation was assayed by 3H-TdR incorporation,and IL-12 secretion in the DC supernatant measured by ELISA.Results:In vitro DC-inducing system IL-10 had an obviously negative effect on the maturation as well as the potentiation to stimulate the T cell proliferation and IL-12 secretion of the immature monocyte-derived DC,and IL-10 could drive monocyte-derived DC differentiate into the macrophages.The negative effect was also correlative to the concentration of the added IL-10;The results also showed that IL-10 hadn′t any negative effect on mature DC induced by sCD40L,but to some extent could reduce the mature DC induced by TNF-? to produce IL-12;Furthermore the inhibitory effect of IL-10 can′t be reversed by adding TNF-? or sCD40L after IL-10 was added to the DC-inducing culture system for three days.Interesting by adding sCD40L not TNF-? to the DC-inducing culture system with IL-10 at the same time can inhibit the negative effect of IL-10 completely.Conclusion:IL-10 is an important biological factor produced in tumor microenvironment for escaping the attack of the immune system by repressing maturation,potentiation to costimulate the T cells and IL-12 secretion of the immature monocyte-derived DC.The reverse effect of TNF-? and sCD40L on IL-10 negative effect on monocyte-derived was different.All together suggested that CD40 signal has important values to obtain the therapeutic DC for the tumor immune intervention.

AIM: To prepare the efficient tumor-DC vaccines, dendritic cells(DC) derived from 6-8 weeks Balb/c mice bone marrow progenitor cells were pulsed by apoptotic SP2/0 tumor cells and induced maturation by SP2/0 tumor lysates supernatants. Then SP2/0 tumor burdening Balb/c mice were immunized by the tumor-DC vaccines to observe the therapeutic effects in vivo .METHODS: Immature DC were derived by recombinant murine GM-CSF and IL-4, then were pulsed by SP2/0 apoptotic cells. Tumor-DC vaccines were stimulated by LPS and SP2/0 tumor lysates supernatants prepared by four cycles repetitive freezing and thawing, respectively. -thymidine incorporation test and standard 4h [ 51 Cr] release assay were used to detect the proliferation and activation of cytotoxic T lymphocytes (CTL) stimulated by DC in vitro . (4-5)?10 5 DC were immunized in the right inguen of SP2/0 tumor burdening Balb/c mice and most mice received three cycles immunization every two weeks. Changes of the tumor and mice life-spans were recorded. RESULTS: In vitro proliferation and activation of CTL induced by the tumor-DC vaccines of tumor lysates supernatants or LPS stimulation group were more powerful than other groups ( P

Objective: To analyze the association between tea drinking during pregnancy and the risk of preterm delivery and abortion,so as to provide basis for prevention of preterm delivery and abortion. Methods: The databases of CNKI,Wanfang,VIP,CBMdisc,PubMed and Web of Science were searched for cohort studies and case-control studies into the association between tea consumption during pregnancy and preterm delivery or abortion until June 30 th,2019. Relative risk（RR）or odds ratio（OR）were used as indicators for the meta-analysis. Results: A total of 1 099 articles were retrieved,14 of them were included in the quantitative study,including 9 cohort studies with 18 295 exposed and 71 890 unexposed individuals and 5 case-control studies with 1 351 cases and 3 059 controls. There was no statistically significant association between tea drinking during pregnancy and preterm birth or abortion（OR/RR=1.08,95%CI：0.99-1.18）. The linear regression model of random effect showed that with the increase of tea consumption during pregnancy,the risk of premature delivery and abortion did not change significantly（OR/RR=1.05，95%CI：0.99-1.11）. There was no publication bias found in Begg's test and Egger's test. Conclusion Drinking tea during pregnancy is not associated with preterm delivery and abortion.

By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene trans- fection technique. Under the induction of cortisol (1 μmol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen Ⅱ and Ⅴ and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard con- centration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that de-coy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.

The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 microl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 microl) were 86614 +/- 13776, 73245 +/- 15414, 61078 +/- 12124, 54657 +/- 10953, 39802 +/- 11308, 32014 +/- 15057 respectively, with the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.
Bone Neoplasms ; metabolism ; pathology ; Dependovirus ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Osteosarcoma ; metabolism ; pathology ; Transfection ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; biosynthesis ; genetics