Objective To knockout Rag2 and IL2rg genes and construct severe combined immunodeficiency mice based on CRISPR/Cas9 technology. Method Design and synthesis of 25 bp sgRNA were made according to the Rag2 and IL2rg sequences in Genbank. After annealing, sgRNA was cloned into pX330 vector. Recombination plasmid Rag2?sgRNA, IL2rg?sgRN and Cas9 were then transcribed into RNA, these RNA were microinjected into zygotes and the zygotes were transplanted into recipient ICR mice. F0 founders were born and mutated F0 founders mated with wild type mice to obtain F1 generation heterozygous mice. Mutated F1 mice were crossed and got F2 generation homozygous mice. Genotype and phenotype of the knockout mice were identified by sequencing, flow cytometry and xenograft model. Results Rag2?sgRNA and IL2rg?sgRNA recombination plasmids were constructed and transcribed into RNA. After microinjection and mat? ing, F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2rg gene, 10 bp or 11 bp deletion;however, there was only one genotype in Rag2 gene, which was 8 bp deletion. Compared with wild?type BALB/c mice, the number of CD3 +, B220 + and NKp46 + cells in peripheral blood of the knockout mice was reduced significantly. After inoculation of human breast cancer cell line SKBR?2HL cells, tumor size in the xenograft mouse model was increased gradually along with time extension. Conclusion CRISPR/Cas9 is an efficient way to mutate Rag2 and IL2rg gene in mice in vivo, leading to aberrant T cells, B cells and NK cells.

Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank .sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C 57BL/6 mice.After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile , real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice .Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas 9.After microinjection, miRNA-29b1 gene-mutated mice were obtained.The sequencing results showed that there were two types of genotype for the mutated mice , one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion.Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly .Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR /Cas9 technology.

Objective To analyse MMACHC mutations for 45 pedigrees with combined methylmalonic aciduria and homocyctinuria by Sanger sequencing, and to discuss the utility of prenatal genetic diagnosis for these pedigrees.Method Peripheral blood was collected from 45 probands and their parents from 2012-2015 in Genetic Counselling Clinic of the First Affiliated Hospital of Zhengzhou University, and the DNA were extracted from the blood.Then the coding sequence of MMACHC gene was amplified by PCR, and the PCR products were further sequenced to detect mutations for each pedigree.For 12 families, chorionic villus sampling was performed on the pregnant women to make prenatal genetic diagnosis.Result There were 14 distinct mutations detected in the 45 pedigrees, and the most frequent mutations are c.609G>A(W203X),c.658-660delAAG(K220del)and c.80A>G (Q27A).Two of those mutations have not been reported before:one is a splicing site mutation c.81+1G>A;while the other is a missense mutation c.665A>G,p.Y222C.Most mutations were found in exon 4.Among the 12 pedigrees who received prenatal diagnosis, 2 fetuses were normal, 7 fetuses were carriers of heterozygous mutation, and the other 3 fetuses were patients with compound heterozygous mutation or homozygous mutation.The couples whose fetuses were normal or carriers continued the gestation, while the couples whose fetuses were patients decided to terminate the pregnancy.After delivery, the outcome of the fetuses was the same as the prenatal diagnose results.Conclusion Two novel mutations of MMACHC were identified and prenatal genetic diagnosis helps to avoid the delivery of combined methylmalonic aciduria and homocyctinuria patients.

The development, application and revision of the clinical practice guideline (CPG) in traditional Chinese medicine (TCM) are a whole thing. However, the development and revision of TCM CPGs have been influenced due to lack of TCM CPGs reporting and feedback channel. Therefore, during the TCM standardization network establish-ment, we studied the application model of TCM CPGs with spontaneous reporting network, in order to provide the ba-sis for further TCM CPGs development and revision.

OBJECTIVETo observe the differences in the clinical efficacy on depression in comparison among the alliance of acupuncture and Chinese herbal medicine, simple herbal medicine and flupentixol and melitracen tablets.

METHODSTwo hundred and twenty-seven cases were randomized into a Chinese medicine group(75 cases), an acupuncture + medication group(78 cases) and a western medicine group(74 cases). In the Chinese medicine group, Shugan Jianwei Anshen Decoction was prescribed, in which Radix Bupleuri and Radix Paeoniae Alba were the monarch herbs, one dose a day. In the acupuncture + medication group, on the basic treatment of Chinese medicine, acupuncture was combined every day at Baihui (GV 20), Sishencong (EX-HN 1), Shenmen (HT 7), Lingdao (HT 4), Daling (PC 7), Laogong (PC 8), Yongquan (KI 1), etc. In the western medicine group, deanxit was taken orally, 1 tablet after getting up in the morning and after lunch respectively. The treatment lasted for 8 weeks in the three groups. The change of Hamilton depression scale (HAMD) score and clinical efficacy were observed before and after treatment.

RESULTSIn 8 weeks of treatment, in the acupuncture + medication group, 19 cases were cured, 35 cases were markedly effective, 14 cases were effective and 10 cases failed, the effective rate was 87.2%; in the western medicine group, 11 cases were cured, 32 cases were markedly effective, 18 cases were effective and 13 cases failed, the effective rate was 82.4%; in the Chinese medicine group, 5 cases were cured, 14 cases were markedly effective, 35 cases were effective and 21 cases failed, the effective rate was 72.0%. HAMD assessment was done in 8 weeks of treatment, which was (5.71 +/- 4.32) scores in the acupuncture + medication group, (6.09 +/- 3.78) scores in the western medicine group and (9.24 +/- 3.49) in the Chinese medicine group, the differences were significant in the Chinese medicine group compared with the western medicine group and the acupuncture + medication group (both P < 0.05). The differences in the scores after treatment were not significant statistically between the acupuncture + medication group and the western medicine group (both P > 0.05), but the cured and markedly effective rate in the acupuncture + medication group was superior to that in the western medicine group (P < 0.05).

CONCLUSIONThe alliance of acupuncture and medication achieves the definite efficacy on depression, characterized as less adverse reactions and better safety.

Acupuncture Therapy ; Adolescent ; Adult ; Aged ; Combined Modality Therapy ; Cooperative Behavior ; Depression ; drug therapy ; psychology ; therapy ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Treatment Outcome ; Young Adult

Objective To screen differentially expressed genes in placentas with hepatitis B virus (HBV)infection and to discuss the molecular mechanism of HBV intrauterine infection.Methods Thirty placenta tissue specimens from HBsAg and HBV DNA positive pregnant women were used as the study group and 30 placenta tissue specimens from normal pregnant women with HBsAg and HBV DNA negativity were served as the control group.The suppression subtractive hybridization(SSH)technique was used.Total RNAs of placenta tissue of the study group were mixed as the tester,and total RNAs of placenta tissue of the control group were mixed as the driver.A subtractive cDNA library was constructed by PCR-selective cDNA subtraction systems.Amplifications of the library were carried out with E.coil strain DH5? by reverse spot hybridization.RT-PCR confirmed that phosphatidylinositol 3-kinase(PI3K)was up-regulated in placenta tissue with HBV infection.Results Colony PCR showed that the clones contained 200-1000 bp inserts. Thirty five clones were confirmed by reverse spot hybridization and analyzed by sequencing and bioinformatics.Thirty three known genes and 2 genes with unknown function were obtained.RT-PCR preliminarily confirmed that PI3K gene was up-regulated in HBV infected placenta.Conclusions The differentially expressed genes in placentas with hepatitis B virus(HBV)infection using SSH technique has been screened out successfully.These differentially expressed genes encoding proteins participating in cell vital metabolism and malformation,and signal conduction-antiapoptosis pathway.This finding brings some new clues for studying the mechanisms of HBV intrauterine infection.

Objective To study the COLIXA3 gene polymorphism of patients with fluorosis and to explore the pathogenesis of COLIXA3 gene in endemic fluorosis.Methods Fifty one cases of patients with drinking-water borne fluorosis were selected as the case group in Xinzhou city,Shanxi province and 28 cases of healthy people were as the control group.Dental fluorosis was detected by Dean method and skeletal fluorosis was examined by X-ray.COLIXA3 of exon 5 gene product of 103 points was amplified by PCR and the gene locus genotype was sequenced.Results Ten cases of mild dental fluorosis,14 cases of moderate dental fluorosis,15 cases of severe dental fluorosis were detected among the 51 patients.The control group was free of dental fluorosis.All the 51 cases of patients with fluorosis had varying degrees of skeletal fluorosis,mainly osteosclerosis lesions,accounting for 86.27％(44/51 ),and mild skeletal fluorosis patients were all osteosclerosis lesions,and osteosclerosis lesions and multiple skeletal lesions were found among moderate and severe skeletal fluorosis patients in the case group,while control group had no skeletal fluorosis.The differences between genotypes of frequency distribution of AA,Aa,aa of COLIXA3 of case and control groups were not statistically significant [96.08％(49/51 ),3.92％(2/51 ),0.00％(0/51) and 96.43％(27/28),3.57％(1/28),0.00％(0/28),x2 =0.94,P ＞ 0.05].ConclusionsCOLIXA3 gene polymorphism is not significantly correlated to fluorosis.

Objective To explore whether different degrees of fluorosis influence the expression of cartilage COLIXA3 protein in fluorosis model rats. Methods Forty male Wistar rats 3 to 4 weeks old were randomly divided into 5 groups according to body mass, and these rats were fed with distilled water containing sodium fluoride(NaF) of 0(control), 25, 50, 100 and 150 mg/L for 6 months, respectively, in order to establish the animal model of drinking water type fluorosis. Pathomorphologieal changes of the osseous tissues of rats were analyzed under light microscope and transmission electron microscope, and the expression of COLIXA3 protein of femur metaphysis was examined by immunohistochemistry. Results HE staining showed different degrees of femoral metaphyseal ossification of cartilage in each experimental group, bone density increased, with sclerotic lesions of skeletal fluorosis. The control group showed no abnormal cartilage. Electron microscopy showed that the experimental groups with varying degrees of cartilage cell swelling, cell matrix fades, 50 mg/L group .showed hyperplasia, and 100,150 mg/L groups were observed with organelles decreased, part of the disintegration of the cartilage cell lacunae, lmmunohistochemical staining of rat chondrocytes COLIXA3 was positive, cytoplasm with brown granules, cartilage COLIXA3 protein expression(23.3 ± 4.5, 41.2 ± 5.6, 26.4 ~ 7.5) in the 25, 50 and 100 mg/L groups enhanced. Compared to the control group (6.1 ± 3.5), the expression of 50 and 100 mg/L groups was significantly increased, and the differences were statistically significant(all P < 0.05). The expression(13.3 ± 4.2)of COLIXA3 protein in 150 mg/L group was decreased compared with the previous three, but is still higher than that of control, and the difference was not statistically significant(P > 0.05). Conclusions There has pathological changes of sclerosing skeletal fluorosis in animal model. Low-dose fluoride promotes while high-dose inhibits cartilage cell proliferation. When fluorine concentration in external environment is too high and with extended exposure to fluoride, direct toxic effects of fluoride on cartilage cells is observed. Fluorine affects and promotes the expression of COLIXA3 protein in cartilage. Low-dose fluoride can promote COLIXA3 protein expression, as the dose increases (over 100 mg/L), the effect decreases.

ObjectiveTo study the relationship between expression of a3 chain of collagen Ⅸ (COLIXA3)mRNA in the population exposed to fluorine and fluorosis,in order to reveal the role of COLIXA3 gene in the pathogenesis of endemic fluorosis.MethodsTwelve cases of mild drinking water-born skeletal fluorosis were selected as case groups in Regiment 123 and 128 of Xinjiang Production and Construction Corps Seven Division,6cases of healthy people living in fluorosis areas for more than 10 years as a internal control group and 6 heathly cases living in non-fluorosis areas for more than 10 years as a external control group.The expression of COLIXA3mRNA of peripheral blood lymphocyte of skeletal fluorosis patients and control groups were determined by using SYBR Green Ⅰ chimeric fluorescent method for real-time quantitative PCR.ResultsThe results of the relative expression of COLIXA3 mRNA of case group,internal control group and external control group were 2.16 ± 0.62,1.06 ± 0.09 and 1.05 ± 0.12,respectively.The COLIXA3 expression in case group was significantly higher than that of the internal control group and the external control group (all P ＜ 0.05),while the difference of COLIXA3expression between the internal control group and the external control group was not significantly different (P ＞0.05).ConclusionsFluorine contributes to the expression of COLIXA3 mRNA in peripheral blood lymphocyte,and the expression is up to 2 times higher than that of the control groups,meaning potential biomarkers.

The RNA-directed DNA Methylation (RdDM) is one type of epigenetic modification which was firstly discovered in plant. RdDM can directly cause DNA modifications of the genome through RNA-DNA interactions. In plant, both of RdDM and mRNA degradation induced by siRNA can silence sequence specific genes through RNA. They play very significant roles in chromosome rearrangement, defence of virus invasion, regulation of gene expression and many processes of plant development. However, the mechanisms of RdDM are still unclear. In this paper the basic characteristics of RdDM were briefly summarized and advances in studies on mechanisms of RdDM were reviewed. These include the kinds of DNA methyltransferases and their functional mechanisms in RdDM, the relationships between DNA methylation and chromatin modification, and important proteins involved in the RdDM process. In plants, RdDM may occur at both the transcriptional and post-transcriptionnal levels, both of which induce gene silencing. Methylation of the target gene promoter correlates with transcriptional gene silencing (TGS) whereas methylation of the coding sequence is associated with post-transcriptional gene silencing (PTGS). RdDM and RNAi all depend on the similar siRNA and enzymes, such as DCL3, RdR2, SDE4 and AGO4. There are at least three kinds of DNA methyltransferases, DRM1/2, MET1 and CMT3, in pants. They can interact with and modifies all cytidines within the DNA regions homologous to RNA sequence. Furthermore, methylation of lysine 9 in Histone H3 can affect the methylation of cytidines.
DNA Methylation ; DNA Modification Methylases ; metabolism ; Gene Silencing ; Plants ; genetics ; metabolism ; Protein Processing, Post-Translational ; RNA ; metabolism