Objective To explore the clinical effect of insulin glargine combined with acarbose in treatment of type 2 diabetes mellitus(T2DM).Methods 189 T2DM patients were randomly divided into combined treatment group and the control group by digital table.Two groups were given the same basic treatment,combined treatment group was given glargine insulin(30R) and acarbose treatment,control group was given insulin glargine(30R) treatment.Clinical efficacy was compared between the two groups.Results After 3 months of treatment,two groups of patients after treatment,FPG and 2h glucose were improved (t =1.987,2.632,2.009,2.687,all P ＜ 0.05).In combined treatment group,2b glucose after the meal [(7.3 ± 2.7)mmol/L] and effective rate(97.9％) were better than the control group[(8.7 ± 4.1) mmol/L,89.4％] (t =2.687,x2 =13.25,all P ＜ 0.05).Conclusion Insulin glargine combined with acarbose in the treatment of T2DM had stable and reliable therapeutic effect.

According to relevant national laws and regulations, practitioner training was included into laboratory animal science teaching reform.By adjusting the training content and teaching method and use of animal models of typical human diseases, the transformation of training mode was realized and improved.By the assessment of basic theory in combination with practical operation, the thinking ability and hands-on skill of the practitioners are much improved. Through classroom instruction, experimental teaching, quality assessment and tracking survey, the evaluating process of the training quality of training teaching is performed.Therefore, the teaching reform of the laboratory animal science based on the training of practitioners is established.

Objective To evaluate the effect of near infrared heptamethine cyanine dye IR-783-mediated specific tumor imaging in spontaneous tumor of dogs .Methods IR-783 was intraperitoneally injected to nude mice models of transplanted tumor in a dose of 5μmol/kg.The metabolic time course of IR-783 was detected by in vivo imager .Based on the results of above observation , IR-783 was injected to dogs with spontaneous tumor in a dose of 1.5μmol/kg.The site of intravenous injection was the hind leg .Tumor and peri-tumoral tissues were removed at 5 days after IR-783-injection for fluorescence imaging , pathology and frozen section fluorescence examinations .Results After i.p.IR-783 injection to nude mice models of transplanted tumor , the transplanted tumor tissues of nude mice had stronger specific fluorescence than normal tissues by imaging at 8 days after injection .After i.v.IR-783 injection to four dogs with spontaneous tumor , the fluorescence signal in the tumor tissues was stronger than that in the normal tissues at 5 days after injection .Conclusions Near infrared fluorescent dye IR-783 could be specifically taken up by tumor tissues , and can be used for specific diagnosis of tumor.It has an important clinical application prospect .

Objective To examine the proliferative effect of synthetic cyclic human cartilage glycoprotein-39 (HCgp39) on T cell of collagen-induced arthritis (CIA) rat,and to explore the role of HCgp39 in rheumatoid arthritis (RA).Methods We established the rat model of the collagen-induced arthritis (CIA).The T lymphocytes were isolated and incubated with HCgp39.Proliferation of T cells was determined by cell counting kit-8.Results Two weeks after the first immunization,T cell response to HCgp39 was more significant in CIA groups than in controls( P＜0.01 ),and the response was associated with disease course ( r =0.732,P＜0.01 ) and anti- HCgp39 antibody ( r =0.460,P＜0.01 ).A strong correlation between T cell proliferation and pannus ( r =-0.516,P＜0.01 ),synovium score ( r =-0.346,P＜0.01 ) was also observed.Besides,the levels of anti- HCgp39 antibody and comp in each CIA group were significantly higher than in controls( P＜0.01 ),and the anti- HCgp39 antibody strongly correlated with disease course( r=0.346,P＜0.01 ) and comp( r =0.235,P＜0.01 ).Conclusion The proliferative response of T cell to HCgp39 was found in the early stage of CIA rat,and the HCgp39 peptide antibody was detected in serum,suggesting that the HCgp39 antigen plays an important role in the pathogenesis of early RA.

Objective To optimize and establish ELISpot assay for CCP/AST which could secrete IFN-γ and IL-4, and explore the role and clinical significance of CCP/AST cells in occurrence and development of RA disease. Methods CCP was used as specific-stimulator with FLAG peptide as a control,the frequencies of positive SFC which could specifically secrete IFN-γ and IL-4 in 64 cases of RA, 64 cases of non-RA autoinunune diseases and 30 cases of healthy individuals were tested by ELISpot technique. The diagnostic value of CCP/AST cells was evaluated in patients with RA disease. Meanwhile, the relationships among the indexes above and patient joint symptoms as well as other laboratory parameters were further analyzed and discussed. Results The results showed that the mediam numbers of IFN-γ-SFC and IL-4-SFC were 39(12-77)/3 x 105 PBMC and 1 (1-3)/3 × 105 PBMC in RA patients, the positive rates were 81.3% and 18. 8% respectively. The median value of IFN-γ-SFC/IL-4-SFC ratio was of 15(5-39), the positive rate was of 78. 1%. Both IFN-γ-SFC and ratio of IFN-γ-SFC/IL-4-SFC were significantly higher than those of non-RA diseases (Z = - 7. 458, - 7. 019, P < 0. 01 ) and healthy control ( Z = - 6. 643, - 5. 760, P <0. 01 ), also both these parameters in RA patients with positive anti-CCP antibody and negative anti-CCP antibody were significantly higher than those patients with systemic lupus erythematosns ( Z = - 6. 573, - 6. 098, - 4. 552, - 4. 726, P < 0. 01 ), ankylosing spondylitis ( Z = - 3. 520, - 3. 326, - 2. 950,-2. 126, P<0. 01 or 0. 05), other autoimmune diseases (Z = -4. 838, -4. 418, - 3. 681, -3. 839,P < 0. 01 ) and healthy controls ( Z = - 6. 553, - 5. 578, - 4. 635, - 4. 163, P < 0. 01 ). Combining IFN-γ-SFC, IL-4-SFC with IFN-γ-SFC/IL-4-SFC for RA diagnosis, the area under curve of receiver operating characteristic( ROCAUC) and Youden index were 0. 910 and 0. 747. The diagnostic sensitivity and specificity were 87. 5% and 87.2%. Positive and negative predictive values were 82. 4% and 91.1%, respectively. Correlation analysis showed that ratio of IFN-γ-SFC/IL-4-SFC was not only significantly correlated with antiCCP antibody(r =0.393, P <0.01), but also correlated with joint symptoms in patients such as with number of joints swelling-pain ( r = 0. 429 , P < 0. 01 ), number of joints damage ( r = 0. 463, P < 0. 01 ),rheumatoid factor (r = 0. 166, P < 0.01) and erythrocyte sedimentation rate (r=0. 199,P<0.05).Conclusions There is widely existence of CCP/AST cells activation and abnormity in RA patients,indicating higher frequency of CCP-specific Th1 cells. These results provides a new experimental evidence for an objective understanding the function of specific cellular immune responses and cytokine network regulation mediated by citrullinated proteins in RA. So, the assay for CCP/AST cells has potential values in RA diagnosis and clinical application.

ObjectiveTo examine the proliferative effect of synthetic C Ⅱ260-272 peptide on T cells of collagen-induced arthritis(CIA) rat,and to explore the role of C Ⅱ 260-272 in RA.MethodsThe rat model of collagen-induced arthritis(CIA) was established.The T lymphocytes were isolated and incubated with C Ⅱ 260-272 peptide.Proliferation of T cells was determined by cell counting kit-8.The data were analyzed with SPSS 17.0.The Mann-Whitney U test was used for proliferation levels comparison between the two groups,and the relationship of cell proliferation with other indicators was analyzed with Spearman's correlation.Antibody levels between the two groups were compared using t test.ResultsThree weeks after the first immunization,T cell response to C Ⅱ 260-272 in the CIA groups [0.963 (0.332-1.628)] was more significant than in controls[0.332 (0.331-0.357)](P＜0.05),and the response was associated with disease course(r=0.624,P＜0.01 ).Strong correlation between T cell proliferation and the antibodies,including antibC Ⅱ antibody and anti-Cit-bC Ⅱ antibody was also observed(P＜0.01 ).Two weeks later,the levels of anti-C Ⅱ peptide antibody in each CIA group were significantly higher than those in the controls(P＜0.01).The antibodies strongly correlated with pannus formation (P＜0.01,P＜0.05).ConclusionThe proliferative response of T cell to C Ⅱ 260-272 peptide can be found in the early stage of CIA rat,and the anti-C Ⅱ peptide antibody could be detected in the serum,which suggeststhat the C Ⅱ peptide may play an important role in activating T/B lymphocytes and causing immune reaction when CIA is induced by C Ⅱ.

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 A novel compound based on near-infrared fluorescence (NIRF) probe was prepared to achieve dynamic monitoring of an inflammatory brain edema model in mice and real-time evaluation of therapeutic effects throughin vivo imaging.Methods The NIRF probe IR-783 was chemically linked with clinical brain edema therapeutic drug furosemide (FSM) to obtain the new compound, IR-783-FSM. The ultraviolet fluorescence properties of the compound were evaluated using an ultraviolet spectrophotometer. The uptake of the compound by mouse macrophage cells RAW 264.7 was detected with in vitro cellular experiments. Its cytotoxicity was evaluated through CCK8 assays. A brain edema model was established in BALB/c mice via intraperitoneal injection of lipopolysaccharide (LPS), confirmed by HE staining and dry-wet weight methods for brain tissues. The mice in the brain edema model were divided into control group, IR-783, and IR-783-FSM treatment groups, receiving intraperitoneal injections of PBS, IR-783, and IR-783-FSM, respectively. Real-time in vivo fluorescence imaging was then performed. The mice in each group were euthanized after 10 hours. Ex vivo brain imaging and dry-wet weight measurements were performed to observe the NIRF imaging characteristics and therapeutic effects of IR-783-FSM on brain edema model.Results The newly synthesized compound, IR-783-FSM, retained the excellent near-infrared fluorescence characteristics of IR-783. It could target mouse macrophages with an IC₅₀ of 48.82 µmol/L. A brain edema model could be successfully constructed with intraperitoneal injection of LPS, with significantly higher brain tissue water content compared to the control group (P<0.01). In vivo imaging showed that IR-783-FSM had a significantly stronger fluorescence signal in the brain edema model than IR-783. Compared to the control group, the brain water content was significantly reduced in the 2, 5, and 8 mmol/L IR-783-FSM treatment groups (P<0.01).Conclusion The newly synthesized NIRF probe IR-783-FSM facilitates dynamic monitoring of brain edema and real-time evaluation of therapeutic effects.

The ideological and political content of the laboratory animal science degree course with the basic task of "cultivating morality and cultivating people" is organically integrated into the teaching system of laboratory animal science. It can have a subtle influence on students' thoughts and behaviors. Combined with the curriculum design and professional characteristics of laboratory animal science, this article discussed the ideological and political elements contained in this course, proposed the forms and methods of integrating ideological and political elements into the curriculum design in each chapter. Additionally, the typical cases and characteristic practices of the organic connection of ideological and political education in the teaching system of laboratory animal science were summarized. Practice has proved that integrating the ideological and political elements into the teaching system of laboratory animal science can enhance teacher's awareness and ability of politics, thus effectively improving the compre-hensive quality of students and enhancing the effectiveness of ideological and political education in laboratory animal science.