IL-36Ra inhibited A1 astrocyte polarization in spinal cord of mice subjected to inflammatory pain / 中国药理学通报

Aim To evaluate the effects of different doses of IL-36Ra on pain behavior and the polarization of spinal A1 astrocytes in mice with inflammatory pain.Methods A total of 32 male C57BL/6 mice were divided into four groups: CFA+Saline group, CFA+IL-36Ra 50 ng group, CFA+IL-36Ra 100 ng group and CFA+IL-36Ra 200 ng group by random grouping.The inflammatory pain model was established by injection of complete Freund's adjuvant(CFA)into the plantar surface of the right hind paw of mice.The drugs were given daily from the 1st day to the 7th day after CFA injection in each group by intrathecal injection.The changes in the mechanical withdrawal threshold(MWT)and the radiant heat stimulating paw withdrawal latency(PWL)of the mice were detected before and 1, 3, 5 and 7 days after the CFA injection.Reverse transcription polymerase chain reaction was used to detect the expression changes of A1 and A2 astrocyte markers after IL-36Ra treatment.Immunohistochemistry was used to test the effect of IL-36Ra on the co-expression level of A1 astrocyte marker C3 and GFAP in the spinal dorsal horn.Results MWT and PWL of the ipsilateral paw significantly decreased after the CFA injection, and IL-36Ra(100 ng, 200 ng)treatment could significantly improve the mechanical allodynia and thermal hyperalgesia of CFA mice.After treatment for 7 days, IL-36Ra 200 ng successfully reversed the increase of GFAP and Lcn2 expression in the spinal cord of CFA mice, which demonstrated IL-36Ra could inhibit the activation of astrocytes.IL-36Ra significantly inhibited the expression of A1 astrocyte maker Serping1, H2-T23 in spinal cord but showed no effects on the expression of A2 astrocytes marker with each dose.Furthermore, IL-36Ra inhibited the expression of C3 within the astrocytes in the spinal dorsal horn of CFA mice.Conclusion IL-36Ra attenuates the inflammatory pain via inhibiting the polarization of A1 reactive astrocytes in the spinal cord of mice with inflammatory pain.

Adenovirus-mediated Expression of B-domain-Deleted Human Coagulation Factor Ⅷ in ADSC of SD Rats / 中国实验血液学杂志

OBJECTIVE@#To transinfect SD adipose tissue-derived stem cell (ADSC) in vitro with a recombinant adenoviral vector containing human B-domain-deleted FVIII (BDDhFⅧ), so as to lay the foundation for the treatment of hemophilia A by using ADSC combined with BDDhFⅧ gene.@*METHODS@#ADSCs were isolated from the inguinal adipose tissue of SD rats and passed to third passage for identification. Third passage ADSCs were transfected in vitro with recombinant adenovirus vector Ad-BDDhFⅧ-GFP. The experiments were divided into Ad-BDDhFⅧ-GFP-transfected ADSCs group (A), Ad-GFP-transfected ADSC group (B), and untransfected ADSC group (C). CCK-8 method was used to detect the proliferation of transfected cells in three groups, and the expression level of hFⅧ antigen in cell supernatant was detected by ELISA. RT-PCR and Western blot respectively were used to detect the mRNA and protein expression of BDDhFⅧ in the three groups after transfection.@*RESULTS@#The growth curve of third passage cells isolated and cultured showed an inverted "S" shap; the flow cytometry detection showed the positive expression of CD29, CD90, CD44, and the negative expression of CD45 in third passage cells. After the adipogenic and osteogenic induction, the cells could transformed to adipogenic and osteogenic directions. CCK-8 detection showed that the proliferation of cells in 3 groups not was influenced. ELISA showed that the expression of hFⅧAg in group A was significantly higher than that in group B and C (P＜0.05). RT-PCR showed that compared with group A, there was no target band in B and C groups, and BDDhFⅧ gene was not expressed. The results in group A were consistent with the length of amplified fragments, and BDDhFⅧ target gene was expressed. Western blot analysis showed that the expression of hFⅧ protein in group A was significantly higher than that in group B and C. (P＜0.05).@*CONCLUSION@#Recombinant adenovirus Ad-BDDhFⅧ-GFP can effectively transfect rat ADSC in vitro, which lays an experimental foundation for gene therapy of hemophilia A.

Application of Nutritional Risk Screening 2002 among COPD inpatients and exploration on relationship NRS 2002 with diagnosis indexes / 肠外与肠内营养

Objective:To explore the potential relationship between nutritional risk evaluated by NRS 2002 and the indexes of physical and biochemical parameters.Methods:From January 2015 to December 2015,218 hospitalized patients with chronic obstructive pulmonary disease (COPD) were randomly selected to assess the nutrition risk by using NRS 2002,and the physical examination indexes and biochemical indicators were also collected.The potential correlations between NRS 2002 score and the physical and biochemical parameters were analyzed.Results:The incidence of nutritional risk (the score of NRS 2002 ≥ 3) was 31.7％ in 218 COPD inpatients.Both age and pulmonary function was significantly related to the incidence of nutritional risk (P ＜ 0.05).The relevance also was found between NRS 2002 score and 2 physical indexes (BMI and FFMI) and 2 biochemical indicators (ALB and PA).Conclusion:The frequency of nutritional risk is very high among hospitalized patients with COPD,especially in old patients.NRS 2002 is a suitable nutritional risk screening tool in COPD patients,but a comprehensive nutrition assessment should combine proper nutritional risk screening tool with physical examination indexes and biochemical indicators in clinical applications.

Chromosomal localization of foreign genes in transgenic mice using dual-color fluorescence in situ hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a highly sensitive and specific dual-color fluorescence in situ hybridization (D-FISH) method used for chromosomal localization of foreign genes in double transgenic mice.</p><p><b>METHODS</b>Two strains of double transgenic mice were used in this experiment, one was integrated with the herpes simplex virus thymidine kinase (HSV-tk) and the enhanced green fluorescence protein (eGFP), the other was with the short hairpin RNA interference(RNAi) and beta(654). Splenic cells cultured in vitro were arrested in metaphase by colchicine and hybridized with digoxigenin-labeled and biotinylated DNA probes, then detected by rhodamine-conjugated avidin and FITC-conjugated anti-digoxigenin.</p><p><b>RESULTS</b>Dual-color fluorescence signals were detected on the same metaphase in both transgenic mice strains. In HSV-tk/eGFP double transgenic mice, strong green fluorescence for HSV-tk and red for eGFP were observed and localized at 2E5-G3 and 8A2-A4 respectively. In beta(654)/RNAi mice, beta(654) was detected as red fluorescence on chromosome 7D3-E2, and RNAi showed random integration on chromosomes. It was detected as green fluorescence on chromosome 12B1 in one mouse, while on 1E2.3-1F and 3A3 in the other.</p><p><b>CONCLUSION</b>Highly sensitive and specific D-FISH method was established using the self-prepared DNA probes, and chromosomal localization of the foreign genes was also performed in combination with G-banding in double transgenic mice. This technology will facilitate the researches in transgenic animals and gene therapy models.</p>