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.

OBJECTIVE To explore the improvement effect of rape pollen on androgenic alopecia mice and its mechanism. METHODS The blank group, model group, positive drug group and administration group were set up, the androgenic alopecia mice model was induced by applying 0.2% testosterone after hair removal. The hair growth rate of mice were observed by using 5% minoxidil as positive drug and 0.4 g·mL-1 rape pollen oil solution as administration group. The hair quality and follicle condition of mice were observed by scanning electron microscope(SEM) and HE staining of skin tissue, respectively. The level of VEGF and bFGF in skin were detected by immunofluorescence staining and Western blotting, while the level of serum sex hormones and reactive oxygen species were detected by ELISA. RESULTS Rape pollen could significantly promote the hair growth in mice and improve the state of mice hair scales compared with model group. Mechanism exploration experiments showed that rape pollen could not promote hair regeneration of mice by regulating hormone levels or anti-oxidative stress. However, rape pollen could increase the expression of bFGF and VEGF related to skin angiogenesis at the modeling site. CONCLUSION Rape pollen can promote hair regeneration in androgenic alopecia mice. Its mechanism may be that it promotes perifollicular vascular regeneration by increasing bFGF and VEGF level.

Objective:To investigate the effect of Chinese reading aloud training (c-RAT) on cognitive function and activities of daily living in patients with vascular cognitive impairment no dementia (VCIND).Methods:From January 2018 to January 2019, totally 80 patients with VCIND in the General Hospital of Xingtai Mining Group were enrolled.Subjects were randomized grouped into c-RAT group ( n=43) and control group ( n=37). The c-RAT group was asked to read aloud the Chinese paper for 5 days a week for 12 weeks.At the time of enrollment and 12 weeks after the intervention, the Montreal cognitive assessment (MoCA), trail making test (TMT), auditory verb learn test (AVLT), digit symbol substitution test (DSST) and modified Barthel index (MBI) were filled out. Results:Compared with control group, the difference of D-value in c-RAT group was statistically significant in MoCA total scores(4.00(2.00), 1.50(0.50), Z=3.012, P=0.003), scores of MoCA-Visual space execution (2.00(1.00), 0.00(2.00), Z=2.787, P=0.008), MoCA-attention (1.00(1.00), 0.00(0.50), Z=2.369, P=0.022), MoCA-language (1.00(1.00), 0.00(0.75), Z=3.049, P=0.000)and MoCA-delayed recall(2.00(2.00), 0.00(1.00), Z=2.043, P=0.014), TMT-A scores (-8.00(23.00), 10.50(30.25), Z=2.120, P=0.039), AVLT scores (1earning)(3.00(2.00), 0.50(0.75), Z=2.266, P=0.039) , AVLT scores (recall)(2.00(1.00), 0.00(1.00), Z=2.974, P=0.003)、AVLT scores (recognition) (2.00(0.00), 0.50(1.50), Z=3.054, P=0.000)and DSST scores ((4.96±0.71), (2.39±0.78), t=2.572, P=0.014), while there were no significant differences in the rest parts of MoCA, TMT-B and MBI( P>0.05). The increased scores in DSST were positively correlated ( r=0.205, P=0.006) with the number of reading aloud tasks finished. Conclusion:C-RAT can improve general cognition, especially in information processing speed, executive function, attention and auditory memory.

The widespread application of next generation sequencing (NGS) in clinical settings has enabled testing, diagnosis, treatment and prevention of genetic diseases. However, many issues have arisen in the meanwhile. One of the most pressing issues is the lack of standards for reporting genetic test results across different service providers. The First Forum on Standards and Specifications for Clinical Genetic Testing was held to address the issue in Shenzhen, China, on October 28, 2017. Participants, including geneticists, clinicians, and representatives of genetic testing service providers, discussed problems of clinical genetic testing services across in China and shared opinions on principles, challenges, and standards for reporting clinical genetic test results. Here we summarize expert opinions presented at the seminar and report the consensus, which will serve as a basis for the development of standards and guidelines for reporting of clinical genetic testing results, in order to promote the standardization and regulation of genetic testing services in China.

Objective To study the prevalence and related risk factors of type 2 diabetes with coronary heart disease(CHD) and cerebral infarction after long-term treatment.Methods Two hundred and eighty-five patients with type 2 diabetes who had a hospitalization interval for four to twelve years between the first and second hospital stays were included in group A.Among them,85 patients (about 4 to 7 years) with a hospitalization interval about 5 years (4 to 7 years) between the first and second hospital stays(group B).100 patients with a hospitalization interval about 10 years (8 to 12 years) between the first and second hospital stays(group C).Blood pressure,blood glucose,HbA1c,blood lipids,myocardial enzymology,electrocardiogram,coronary angiography,cranial CT or MRI were tecorded.The prevalence of CHD and cerebral infarction were compare,regression analysis was conducted between coronary heart disease,cerebral infarction and the risk factors.Results DBP,FBG,PBG2h,TC,LDL-C of the second hospitalization were significantly lower than those of the first hospitalization.The morbidity of CHD and cerebral infarction increased along with the extension of the course.The morbidity of cerebral infarction in CHD patients were significantly higher than those in non-CHD patients with the interval about 10 years.Logistic regression analysis proved that age was risk factor and HDL-C was protective factor of CHD,SBP,FBG were risk factors of cerebral infarction.Conclusion The morbidity of CHD and cerebral infarction increased along with the extension of the course and there were significantly relationship between CHD and cerebral infarction in T2DM patients after about 10 years treatment.

Objective To investigate the type distribution of urogenital Chlamydia trachomatis(Ct)among STD clinic outpatients in Guangxi Zhuang Autonomous Region, and to estimate the prevalence of Ct infection among the patients during posttreatment follow?up. Methods Urethral and cervical swabs were collected from male and female outpatients with confirmed urogenital Ct infection, respectively, in Institute of Dermatology of Guangxi Zhuang Autonomous Region. The patients with positive results in preliminary screening tests were followed up after treatment, and specimens were collected at follow?up visits. General and clinical information was also obtained from these patients. DNA was extracted from these samples by using the QIAxtractor instrument. Nested PCR was performed to amplify the major outer membrane protein A(ompA)gene for ompA typing, and to amplify CT046(hctB), CT058, CT144, CT172 and CT682 (pbpB) genes for high?resolution multilocus sequence typing (hr?MLST). Then, PCR products were sequenced, and ompA and MLST types of Ct were determined by sequence alignment and MLST analysis, respectively. The obtained MLST sequence types (STs) were compared with those from an Italian population by using the BioNumerics7 software, and a minimum spanning tree(MST)was generated. Results Totally, 44 and 6 Ct?positive specimens were collected at first visits and follow?up visits respectively. Among the 50 specimens, 42 underwent successful ompA typing and hr?MLST, and 7 ompA genotypes and 15 hr?MLST STs were identified, including 3 first reported STs. The distribution of STs of Ct isolates from Guangxi Zhuang Autonomous Region was significantly different from that from the Italian population. Among the 6 followed patients with posttreatment Ct infection, 3 were confirmed to be reinfected with Ct, and the other 3 failed to be diagnosed because of unsuccessful genotyping. Conclusion The genotypes of Ct strains isolated from STD clinic outpatients in Guangxi Autonomous Region were characteristic, and Ct reinfection occurred in some patients during follow?up.

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 observe effects of different extracts of Jianpi Huogu Formula (JPHGF) on proliferation and differentiation of bone marrow mesenchymal stem cell (BMSCs). Methods Whole bone marrow adherent was used to screen, culture, and isolate BMSCs. Extracts from different parts (water, chloroform, ethyl acetate and n-butanol parts) of JPHGF were administrated for a certain time. MTS was used to detent cell proliferation;ALP staining was used to detect ALP activity;ARS staining was used to detect the formation of calcium nodules;oil red O staining was used to detect fat cell formation. Results Extracts from different parts of JPHGF could promote cell proliferation of BMSCs in different levels, followed by its strength in water, chloroform, ethyl acetate, and n-butanol parts;ALP staining results showed that the intensity of ALP expression of the order is water, acetic acid ethyl, chloroform and n-butanol parts;in promoting the formation of calcium nodules, ARS staining results showed that its intensity were water, chloroform, ethyl acetate, and n-butanol parts;oil red O staining results showed that inhibition intensity of fat cells interaction strength was formed from ethyl acetate, water, chloroform to n-butanol parts. Conclusion Extracts from different parts of JPHGF have different effects on BMSCs proliferation and differentiation. Water extraction has the strongest osteogenic differentiation and proliferation, and ethyl acetate has the best effect on the inhibition of cell formation.

Objective To investigate the clinical features and effective treatment of patients with severe type A H1N1 flu in Wenzhou. Methods The clinical data of 42 hospitalized patients with severe type A H1N1 flu were analyzed and the clinical features were summarized. Results A total of 42 patients with severe type A H1N1 flu all began with fever and cough. The symptoms of expectoration, pharyngalgia, chilly accounted for 92. 9%, 90. 5% and 42. 9%, respectively. The peripheral leucocyte counts were normal or reduced. C-reactive protein and erythrocyte sedimentation rate levels both increased in 30 patients (71.4%). About 95.2% (40/42) patients had changes of pulmonary imaging. All of the patients were treated with oseltamivir and effective antibiotic drugs as well as symptomatic management. No patients was treated with glucocorticoid. The patients with underlying diseases were given proper treatment. Three cases were treated with antifungal therapy and 3 pregnant patients were timely terminated of pregnancy. Conclusions Severe type A H1N1 flu progresses rapidly and the lower respiratory tract is involved soon after onset. Therefore, the patient should be diagnosed early and treated promptly after presenting fever, which will lead to good prognosis.

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.