Objective To explore in vitro mesenchymal stem cell (MSC) transfection of lentiviral vector mediated calcitonin gene‐related peptide(CGRP) gene and its effects on biological properties of MSC .Methods MSC were isolated ,cultured and identi‐fied .MSC were infected by lentivirus encoding recombinant enhanced green fluorescent protein (EGFP) gene and CGRP (Lv‐EG‐FP‐CGRP) .The transfection efficiency was determined by the inverted fluorescence microscope and flow cytometry .The expression levels of CGRP were detected in CGRP‐modified MSC by using real‐time PCR ,immunocytochemistry and enzyme‐linked immu‐nosorbent assay (ELISA) .The proliferation ,aging and differentiation ability of MSC were evaluated by MTT ,β‐galactosidase stai‐ning and inducing differentiation respectively .Results After 48 h of MSC transfection by Lv‐EGFP‐CGRP ,EGFP/CGRP could be expressed stably .When multiplicity of infection (MOI) was 30 ,the transfection efficiency reached more than 80% .Compared with the MSC group and the MSC‐EGFP group ,the mRNA and protein expression levels of CGRP in CGRP‐modified MSC(MSC‐CGRP group) were markedly increased(all P<0 .01) .The results of MTT ,β‐galactosidase staining and inducing differentiation assay dem‐onstrated that the transfected CGRP basically had no effect on the proliferation ,aging and endotheliocyte differerntiation of MSC . Conclusion MSC is a kind of ideal genetic vector cell ,which can serve as the target cell of CGRP gene transduction for the applica‐tion of gene therapy and lays the foundation for follow‐up in vitro and vivo experiments .

Objective To observe the effect of adenovirus- receptor activity modifying protein-1 (RAMP1) on nuclear factor(NF κB) and myocardial fibrosis in heart of rabbit with myocardial infarction. Methods Myocardial infarction (MI) models were developed in 54 rabbits and they were randomly divided into RAMP1 group,EGFP group and control group according to whether pAd2-EGFP-RAMP1,pAd2-EGFP or PBS was injected into infarction region and its border.At 7 d,14 d and 28 d after injection,Left ventricular function indices such as LVEF,LVSd,LVDd and LVFS were estimated by echocardiogram,the expression of NF-κB in myocardium was detected by Western blot,the plasma level of TNF-α was measured by ELISA,and fibrosis and collagen content was measured by Masson stain. Results At 7 d after adenovirus injection,the expression of RAMP1was significantly increased in RAMP1 group (67.33 ± 3.97)％ as compared to EGFP group(20.59 ±3.26) ％ and PBS group ( 23.80 ± 5.08) ％ ( P ＜ 0.05 ).The expression of NF κB was decreased in RAMP1 group ( 26.54 ± 5.13 ) ％ versus EGFP group (62.60 ± 6.18) ％ and PBS group (62.95 ±5.17)％ (P＜0.05).The plasma level of TNF-α was lower in RAMP1 group than in EGFP group and in PBS group at different time[7 d:( 136.74 ± 5.42) μg/L vs.( 196.97 ± 14.17) μg/L,(203.67 ±13.90)μg/L; 14 d:( 154.51 ± 13.61 )]μg/L vs.( 112.22±6.74 )μg/L,(160.46± 14.27)μg/L ;28 d;(51.10± 5.62)μg/L vs.( 95.55 ± 9.94 )μg/L,( 98.96 ± 12.68) μg/L,all P＜ 0.05].The collagen content was reduced in RAMP1 group as compared with EGFP group and PBS group at 14 d and 28 d [14 d:(7.10±0.98)％ vs.(19.52±2.32)％,(17.91±0.96)％ ;28 d:(17.04±2.44)vs,(34.10±5.59) ％,(33.98±4.33)％,all P＜0.01].At 28 d after infarction,the infarct size was decreased in RAMP1 group (26.54 ± 5.13) ％ compared with EGFP group (32.20 ± 3.73) ％ and control group (35.58±2.65) ％ (P＜0.01),and better heart function appeared in RAMP1 group. Conclusions The high-expression of RAMP1 could decrease the collagen deposition and fibrosis in the border of infarction and improve heart function through lower expression of NF-κB and decreasing the plasma level of TNF-α.

Objective To explore the effect of transplantation of mesenchymal stem cells (MSCs) transfected with the human receptor activity modifying protein 1 (hRAMP1) gene on proliferation and apoptosis of vascular smooth muscle cells (VSMCs) after carotid balloon angioplasty was performed in rabbits with carotid atherosclerosis.Methods Density gradient centrifugation and adherent culture were carried out to obtain MSCs,which were then transinfected with an adenovirus vector carrying the hRAMP1 gene or an empty adenovirus vector.A rabbit model of atherosclerotic stenosis and balloon angioplasty was successfully established.Results were randomly divided into three groups:the hRAMP1-MSCs group,theadipose-derived MSCs (Ad-MSCs) group and the control group.MSCs were transinfected with Ad-EGFP-hRAMP1,Ad-EGFP or PBS by transplantation into the injured carotid arteries.Homing and differentiation were assessed with MSCs harvested at 7 d.With MSCs collected at 28 d,Western blotting was used to measure the expression of the hRAMP1 target gene in the carotid artery; the neointima and media area in the injured carotid arteries were estimated; carotid artery morphology was examined with H&E staining; and the proliferation and apoptosis of VSMCs were determined by immunohistochemistry and TUNEL.Results The expression of CD31 and EGFP was found in proliferating neointima lesions at 7d in the hRAMP1-MSCs group and the Ad-MSCs group.At 28d of MSC transplantation,the level of RAMP1 significantly increased in the hRAMP1-MSCs group,compared with the Ad-MSCs and control groups [(63.0±4.9) vs.(28.3±2.5) and (27.2±7.2),all P＜0.05],but there was no differencein the RAMP1 level between the Ad MSCs group and the control group (P＞0.05).Positive expression of the α-smooth muscle antibody (α-SMA) was found in all three groups at 28 d of MSC transplantation.The thickness of the hyperplastic neointima significantly decreased in the hRAMP1-MSCs group,compared with the other two groups (P＜0.05),and was lower in the Ad-MSCs group than in the control group (P＜0.05).The expression of proliferating cell nuclear antigen (PCNA) was lower in the hRAMP1-MSCs group than in the Ad-MSCs and control groups at 28d of MSC transplantation (P ＜0.05),while the PCNA level was lower in the Ad-MSCs group than in the control group (P＜ 0.05).The VSMC apoptosis rate significantly increased in the hRAMP1-MSCs group,compared with the Ad MSCs and control groups (P＜0.05),and was the lowest in the control group (P＜0.05).Conclusions Gene-modified stem cell therapy can effectively inhibit vascular intimal hyperplasia,thereby reducing restenosis after angioplasty.

Objective: To investigate whether microRNA(miR)-214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase-1. Methods: H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37 ℃ with 5%CO₂, repeat passage was made after cell density reached about 80%, The 5^th to 8^th generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR-214 on pyroptosis and caspase-1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimic-negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR-214 mimic-negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti-pyroptosis effect of miR-214 was mediated by targeted inhibition on caspase-1, cells overexpressing caspase-1 were used in the rescue experiment. The cells overexpressing caspase-1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimics+hyperglycosis+recombinant adenovirus (Ad-caspase-1-EGFP) group with caspase-1 gene and EGFP green fluorescent protein expression (mimics+HG+Ad-caspase-1-EGFP group, H9c2 cells were transfected with caspase-1-green fluorescent protein-carrying adenovirus for 48 hours, followed by transfection of miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR-214 mimics+HG+Ad-EGFP empty virus group (mimics+HG+Ad-EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA-214 and caspase-1 in cells were detected by real-time quantitative PCR. The expression and localization of caspase-1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase-1, cleaved caspase-1, NLRP3 and ACS with β-actin as internal reference. The secretion of IL-1β and IL-18 in cell culture medium was detected by ELISA. The correlation between miR-214 and caspase-1 was detected by double luciferase reporter gene. Results: (1) The mRNA expression levels of miR-214 and caspase-1 in each group: the mRNA expressions of miR-214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR-214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase-1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase-1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase-1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL-1β and IL-18 in the cell culture medium of each group: the content of IL-1β and IL-18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL-1β and IL-18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR-214 and caspase-1: miR-214 specifically binds to caspase-1 3 ′UTR. Meanwhile, Western blot results showed that cleaved caspase-1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase-1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase-1 expression among groups (P>0.05). (6) The expression levels of procaspase-1, cleaved caspase-1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase-1 in each group (P>0.05). Cleaved caspase-1, ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase-1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+ Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL-1β and IL-18 in rescue experiment: the secretions of IL-1β and IL-18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). Conclusion miR-214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase-1.

The thyroid function test is universally used for the evaluation of thyroid function. However, there are factors other than endocrine diseases, such as special physiological conditions, interference in laboratory measurement, or disorders in other systems that should be taken into consideration. When encountering thyroid function test results that are not in line with clinical manifestation, a comprehensive evaluation is essential for a correct and timely diagnosis. We presented a case of a significant rise in serum total triiodothyronine(TT 3) secondary to multiple myeloma. Through case studies and literature review, we intended to provide clinicians with experience for better interpretation of abnormal thyroid function test results.