Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

AIM:To explore the role of stromal interaction molecule 1(STIM1)in the migration,invasion and angiogenesis of salivary adenoid cystic carcinoma(SACC),as well as its molecular mechanism.METHODS:Immu-nohistochemistry and Western blot were used to detect the expression level of STIM1 in human SACC tumor tissues and ad-jacent normal tissues.The TCGA database was analyzed to investigate the relationship between STIM1 expression and sur-vival in patients.A SACC-83 cell line with stable STIM1 overexpression(STIM1-OE)was established and divided into two groups:blank control(STIM1-Vec)group and STIM1-OE group.A nude mouse subcutaneous xenograft tumor model(n=6)was used to detect SACC growth.Transwell chamber assay,scratch test and dorsal root ganglion model were ap-plied to assess SACC migration and invasion.Immunohistochemistry was performed to detect the expression of CD34 and STIM1 in tumor tissues of nude mice.A nude mouse Matrigel plug model and human umbilical vein endothelial cell(HUVEC)tube formation assay were used to evaluate angiogenesis.Enzyme linked immunosorbent assay was employed to detect the levels of vascular endothelial growth factor(VEGF)and epidermal growth factor(EGF)in cell culture supernatants.RT-qPCR and Western blot were performed to detect the mRNA and protein expression levels of c-Myc,ribosomal protein L35(RPL35),ribosomal protein SA(RPSA),mitochondrial ribosomal protein L11(RPL11)and FAU ubiquitin like and ribo-somal protein S30 fusion(FAU).The STIM1-siRNA and RPL35-siRNA were transfected into SACC-83 cells,and the mi-gration,invasion and angiogenesis abilities of the cells were detected using the same methods as above.RESULTS:STIM1 was highly expressed in SACC,and the patients with high STIM1 expression had shorter survival time.In vivo,compared with STIM1-Vec group,STIM1-OE promoted tumor growth.In vitro,the number of migrating and invading SACC cells in STIM1-OE group was significantly increased,and the nerve invasion ability was also significantly en-hanced.Conversely,STIM1-siRNA significantly reduced the migration and invasion abilities of SACC cells.Additional-ly,STIM1-OE significantly promoted the expression of the vascular marker CD34,the secretion of VEGF and EGF,and the results of Matrigel plug and HUVEC tube formation assays indicated that STIM1-OE significantly promoted angiogene-sis.Silencing of RPL35 significantly inhibited SACC migration,invasion,and angiogenesis.RT-qPCR and Western blot results showed that the mRNA levels of RPL35,RPSA,MRPL11 and FAU were significantly increased(P<0.05),and the protein expression levels of STIM1,c-Myc and RPL35 were significantly increased(P<0.01).CONCLUSION:STIM1 drive the migration,invasion and angiogenesis of SACC by activating c-Myc/RPL35-mediated ribosome pathway.

AIM:To explore the role of stromal interaction molecule 1(STIM1)in the migration,invasion and angiogenesis of salivary adenoid cystic carcinoma(SACC),as well as its molecular mechanism.METHODS:Immu-nohistochemistry and Western blot were used to detect the expression level of STIM1 in human SACC tumor tissues and ad-jacent normal tissues.The TCGA database was analyzed to investigate the relationship between STIM1 expression and sur-vival in patients.A SACC-83 cell line with stable STIM1 overexpression(STIM1-OE)was established and divided into two groups:blank control(STIM1-Vec)group and STIM1-OE group.A nude mouse subcutaneous xenograft tumor model(n=6)was used to detect SACC growth.Transwell chamber assay,scratch test and dorsal root ganglion model were ap-plied to assess SACC migration and invasion.Immunohistochemistry was performed to detect the expression of CD34 and STIM1 in tumor tissues of nude mice.A nude mouse Matrigel plug model and human umbilical vein endothelial cell(HUVEC)tube formation assay were used to evaluate angiogenesis.Enzyme linked immunosorbent assay was employed to detect the levels of vascular endothelial growth factor(VEGF)and epidermal growth factor(EGF)in cell culture supernatants.RT-qPCR and Western blot were performed to detect the mRNA and protein expression levels of c-Myc,ribosomal protein L35(RPL35),ribosomal protein SA(RPSA),mitochondrial ribosomal protein L11(RPL11)and FAU ubiquitin like and ribo-somal protein S30 fusion(FAU).The STIM1-siRNA and RPL35-siRNA were transfected into SACC-83 cells,and the mi-gration,invasion and angiogenesis abilities of the cells were detected using the same methods as above.RESULTS:STIM1 was highly expressed in SACC,and the patients with high STIM1 expression had shorter survival time.In vivo,compared with STIM1-Vec group,STIM1-OE promoted tumor growth.In vitro,the number of migrating and invading SACC cells in STIM1-OE group was significantly increased,and the nerve invasion ability was also significantly en-hanced.Conversely,STIM1-siRNA significantly reduced the migration and invasion abilities of SACC cells.Additional-ly,STIM1-OE significantly promoted the expression of the vascular marker CD34,the secretion of VEGF and EGF,and the results of Matrigel plug and HUVEC tube formation assays indicated that STIM1-OE significantly promoted angiogene-sis.Silencing of RPL35 significantly inhibited SACC migration,invasion,and angiogenesis.RT-qPCR and Western blot results showed that the mRNA levels of RPL35,RPSA,MRPL11 and FAU were significantly increased(P<0.05),and the protein expression levels of STIM1,c-Myc and RPL35 were significantly increased(P<0.01).CONCLUSION:STIM1 drive the migration,invasion and angiogenesis of SACC by activating c-Myc/RPL35-mediated ribosome pathway.

This study was aimed to develop in-line monitoring of extraction process of Lonicerajaponica in the extraction process trajectory of Re-Du-Ning (RDN) injection.Several batches of near-infrared (NIR) spectrum dates were collected and multivariate statistical process control (MSPC) method was in conjunction with.The results showed that using score,Hotelling T2 and DModX control chart,various normal and abnormal behaviors of the test batches were detected in time by comparison with the extraction process trajectory.It was concluded that the process trajectory for in-line quality control based on NIR spectrum dates and MSPC could indicate the changes of process.It was a feasible technology tool of the total process quality control during traditional Chinese medicine (TCM) manufacturing process.

Objective To observe the inhibitory effect of quercetin on the biofilm formation of Streptococcus mutans(Sm),to preliminarily reveal the possible underlying mechanisms,and to evaluate the cytotoxicity of quercetion to human dental pulp cells so as to provide the theoretical basis for the application of quercetin in oral biomaterials.Methods Quercetin storage solution was diluted to 0,3.125,6.25,12.5,25,50,100,200,400 and 800 mg/L,and added into Sm medium for 4 h and 24 h,crystal violet staining was used to evaluate the biofilm volume.In subsequent detections,three groups were set:control(0 mg/L),200 mg/L quercetin and 400 mg/L quercetin.Confocal laser scanning microscopy was used to observe the morphology of the biofilm;qPCR for gtfB,gtfC,comD,comE,and luxS were assessed to preliminarily investigate the mechanisms.Finally,the methyl thiazolyl tetrazolium(MTT) test using human dental pulp cells was used to investigate cytotoxicity.Results Quercetin could significantly inhibit up to (86.16±0.45)％ of the biofilm formation of Sm (Compared with the control group P=0.00) and effectively removed (43.04±0.53)％ of the mature biofilm(Compared with the control group P=0.00).Confocal laser scanning microscopy photographs showed that after co-incubated for 24 h,the dense biofilm structures of the experimental group were destroyed by quercetin both at 200 mg/L and 400 mg/L.Quercetin suppressedover 50％ of the expression of gtfB,gtfC,comD,comE(compared with the control group P＜0.05) and promoted the expression of luxS up to 2.18 ±0.24 and 2.84±0.26 after 4 h and 24 h,respectively(compared with the control group P＜0.05).Quercetin also exhibited acceptable compatibility for human dental pulp cells.Conclusions Quercetin could effectively reduce the biofilm formation of Sm by inhibiting the expression of the related genes,and exhibited no cytotoxicity for human dental pulp cells.Quercetin has good potential to be applied in oral biological materials.

Objective To evaluate the surgical technique and the clinical effect of applying free superficial palmar branch of the radial artery flap to repair soft tissue defect of finger pulp.Methods From June,2011 to December,2013,12 cases of soft tissue defect of finger pulp and bone exposed were treated with free superficial palmar branch of the radial artery flap.The flap was designed from the proximal end of palmar wrist and the donor site was sutured directly.The size of the harvested flaps was between 2.5 cm × 1.0 cm and 6.2 cm × 3.8 cm,and the sensation of the flap was reconstructed via median nerve cutaneous branch.Results All transfering flaps survived and all cases were followed up for 4 to 17 months.The donor site got primary healing with a straight car.The appearance and texture of the flaps were satisfactory.2-point discrimination ranged from 6 to 11 mm.The pain sensation,warmth sensation and touch sensation of the flap got better.Conclusion The free superficial palmar branch of the radial artery flap is easy to harvest and anastomose,which is masked and a small incision for the donor site.The appearance and sensation of the flap which has sensory nerve branched for sensory reconstruction is satisfactory.It is an ideal method for the repairment of finger pulp defects.

Objective To compare the effect of 4 different cold and hot properties of traditional Chinese medicine (TCM) on body temperature and related factors of yeast induced fever rats,and discuss the thermoregulatory mechanism of cold and hot properties of TCM.Methods 108 male SD rats were randomly divided into a normal group,a yeast-induced group,a R.palmatum treated group,a C.chinensis treated group,a Euodia ruticarpa treated group,and a Alpinia officinarum Hance treated group,with 18 rats in each group.Pyrexia model was induced by injecting yeast suspension subcutaneously on rat.At the 4h,8h and 12h after injection of yeast,the rats were sacrificed,and the blood and hypothalamus were collected.The levels of prostaglandin E2 (PGE2),cyclic adenosine 3',5'-monophosphate (cAMP) and arginine vasopressin (AVP) in hypothalamus and plasma were detected by ELISA assay.Results At the 4h after injection of yeast,the temperature of rats in the model group began to rise,and it reached the peak at 8h,while RheumpalmatumL and Coptis chinensis could significantly reduce the body temperature of yeast-induced rat (P＜ 0.01 or P＜ 0.05).At 8h,the levels of PGE2 and cAMP in hypothalamus increased significantly [respectively (31.55 ± 9.88) pg/mg and (0.17±0.03) pmol/mg] compared with the normal group,while the level of AVP (0.14±0.02) pmol/ml in plasma reduced (P＜0.05).Compared with model group,at 8h RheumpalmatumL and Coptis chinensis could significantly lowered PGE2 [respectively (113.65± 18.60) pg/mg and (127.72 ± 15.75) pg/mg,P＜ 0.01 or P＜0.05],and cAMP [respectively (0.69±0.08) pmol/mg and (0.74±0.10) pmol/mg,P＜0.05] in hypothalamus,and increased AVP levels [respectively (1.08 ± 0.12) pmol/ml and (0.91 ±0.01) pmol/ml,P＜0.05 or P＜0.01] in plasma.Euodia ruticarpa and Alpinia officinarum had no significant effect on both body temperature and the levels of inflammatory factors.Conclusion The two cold property traditional Chinese medicines,R.palmatum and C.chinensis,could significantly reduced the body temperature of yeast-induced rats,which may be related to its effective regulation on levels of PGE2 and cAMP in hypothalamus and AVP in plasma,however,the two hot property traditional Chinese medicine,Euodia ruticarpa and Alpinia officinarum Hance,had no related effects.

To observe the influence of the placental apoptosis on the expression of Bax,Bcl-2, Fas, FasLand TNF-α during the second trimester of pregnancy, mice of experimental group were intraperitoneal injected with 100 purified Toxoplasma gondii tachyzoites added in 0.2mL of PBS, while those of the control group were injected with 0.2 mL of sterile PBS (0.01 mol/L, pH 7.4) in the 8-th day of pregnancy. During the 12, 14, 16 and 18-th days of pregnancy, 5 mice both in experimental and control group were randomly killed and the expression levels of the apoptosis-related proteins Bax, Bcl-2, Fas, FasL and TNF-α in the placental tissues were determined by means of immunohistochemical methods. It was showed that the apoptosis-related protein expressed both in villus and decidua of the placenta, most of which were expressed in syneytiotrophoblast (ST). The positive cells with expression of Bax, Fas, FasL and TNF-α increased along with the increase of the pregnant days in both the experimental group and the control group, and the positive cells with expression of Bcl-2 decreased along with the increase of the pregnant days. It was also demonstrated that the positive cells with expression of Bax, Fas, FasL and TNF-α of the experimental group showed a higher percentage of expression than that of the control group on the same pregnant days, but the positive cells with Bcl-2 expression of the experimental group were fewer than that of the control group. It is concluded that the expression of apoptosis-related protein Bax, Bcl-2, Fas, FasL and TNF-α in the placenta were altered when the pregnant mice were infected with Toxoplasma gondii during the second trimester, which may induce the apoptosis through the endogenic and ectogenic pathway.

Objective:To check the effect of immunosuppressants as adjuvants for DNA vaccine to prevent autoimmune disease.Methotis:Three immunosuppressants,Cyclosporin A(CsA),Tacrolimus(FK506)and Mycophenolate Mofetil(MMF)Were combined with DNA vaccine of Multiple Sclerosis(MS)and injected intramuscuarly.The splenocytes of immunized mice were checked for regulatory T cells(Treg),T cell proliferation and maturation of dendritic cells(Dcs)as well as release of intereleukin 10(IL-10).The immunized mice were induced as EAE animal model to check the preventive effect of FK506 as adjuvant.Results:More Treg cells Were induced in the mice immunized with FK506 and DNA vaccine and also T cell proliferation Was suppressed.DCs maturation Was blocked and high level of IL-10 in DCs Was induced in mice treated with FK506 and DNA vaccine.The clinic scores of EAE were significantly decreased in mice treated with FK506 and DNA vaccine compared with other control groups.Conclusion:FK506 as adjuvant of DNA vaccine can stimulate Treg cells and immune tolerance,then prevent from the autoimmune disease.