Objective: Pocket infection in patients after total removal of implanted pacemaker has many problems for their electronic system; our research intends to explore the feasibility of conservatively treating such infection and retain the electronic system. Methods: A total of 4 patients with pacemaker pocket infection in our hospital from 2015-01 to 2016-02 were studied. Thorough debridement and disinfection were conducted in infected pockets and devices, meanwhile vacuum sealing drainage was applied. Electrode wire was kept and intravenous antibiotics were given for (7-10) days after the operation in all patients. Results: The average time of infection occurred at 14.75 months after operation with the type of isolated pacemaker pocket infection. Pocket vacuum suction drainage was performed, with the mean of 7.25 (5-10) months follow-up observation, infection was disappeared and the patients had good wound healing. Conclusion: With thorough debridement of infected pocket, rational treatment of residual electronic system and vacuum sealing drainage, the infection might be effectively controlled for complete recovery without lead extraction in relevant patients.

AIM:To investigate the effect of calcitonin gene-related peptide (CGRP)transfection into c-kitpos cardiac stem cells (c-kit+CSCs) on the cell viability.METHODS: Under the sterile condition, the auricles of SD rats were taken out , and then c-kit +CSCs were collected through enzyme digestion and immunomagnetic bead separation (MACS).The cells were identified by flow cytometry .c-kit +CSCs were transfected with enhanced green fluorescent pro-tein CGRP lentiviral vector ( Lv-EGFP-CGRP) or enhanced green fluorescent protein lentiviral vector ( Lv-EGFP).The cells were randomly divided into Lv-EGFP-CGRP-CSCs group , Lv-EGFP-CSCs group and CSCs group .The transfection was observed under the fluorescence microscope .The transfection efficiency was detected by flow cytometry .The CGRP protein secretion in the cell culture supernatants was detected by ELISA .The viability of c-kit+CSCs transfected with Lv-EGFP-CGRP or Lv-EGFP was measured by CCK-8 assay.RESULTS:c-kit+CSCs were isolated and cultured successfully .The expression positive rate of c-kit was 91.0%and the expression positive rates of CD 45 and CD34 were 4.5% and 4.0%, respectively.After transfected with lentivirus for 48 h, the stable fluorescence in c-kit +CSCs was observed under fluores-cence microscope .The transfection efficiency were 80%when MOI was 20.The level of CGRP was significantly increased in Lv-ECFP-CGRP-CSCs group compared with Lv-EGFP-CSCs group and CSCs group (P<0.05).Meanwhile, transfection with lentiviral vector in each group did not affect the viability of c-kit+CSCs.CONCLUSION:Transfection of Lv-EGFP-CGRP into c-kit+CSCs was successful .The secretion of CGRP was found in the transfected c-kit+CSCs and the viability was not changed after transfection .CGRP-modified c-kit+CSCs may play a role in treating myocardial infarction .

Objective To study the effect of the recombinant Lentivirus containing calcitonin gene related peptide (CGRP) gene on cells biological activity and differentiation of rat bone marrow stem cells(MSCs).Methods Rat MSCs were isolated and cultured by granulocytes adherent.MSCs were transfected with Lenti-EGFP CGRP(MSCsCGRP+/+ group),While MSCs were transfected with Lenti-EGFP as control group.Cell transfection rate was detected by flow cytometry,protein secretion in the above-mentioned MSCsCGRP+ + supernatant was detected using ELISA method.Cells surface markers weare detected by flow cytometry and immunohistochemistry.Trypan blue was used to examin the survive rate,β galactosidase staining was used to examin aging of MSCs transfection,and MTT was used to examine cell vitality.Results At first day after transfecting with Lenti-EGFP-CGRP,fluorescence was not observed by fluorescence microscope,but a small amount of CGRP protein was detected by ELISA in MSCsCGRP+/+ group,at 3 days and 4 days after transfecting with MSCs,strong fluorescence was observed by fluorescence microscope (the cell transfection rates were 77.87％ and 79.58％).The CGRP expression was significantly higher in MSCsCGRP+ + group than in control group [(19.53±0.50) pg/ml vs.(3.12±0.00) pg/ml,t=48.964,P＜0.01].At three days after transfection with MSCs,CD29 and CD90 expression were significantly higher,as compared with control group,CD31 expression was increased in MSCsCGRP+ /+ group.Seven days after transfection with MSCs,CD31 expression was significantly increased in MSCsCGRP+ + group,vWF expression was significantly increased in MSCsCGRP+ + group after MSCs were transfected with LentiEGFP CGRP for 14 days,but a SMA expression was decreased in MSCsCGRP+ +group.At 3 days and 7 days after transfection with Lenti-EGFP-CGRP,the proliferation,survive and aging showed no difference in MSCsCGRP+/+group and in control group (the proliferation of cell:t=0.253,0.290the survive of cell t=-0.307,0.690,all P＞0.05).At 14 days after transfection with Lenti-EGFP-CGRP,aging of cell were decreased in MSCsCGRP+ + group as compared with control group (t=2.446,P＜ 0.05).Conclusions After MSCs are transfected with Lenti EGFP-CGRP,biological characteristics of MSCs has no significant effects,there is still proliferation and differentiation activity.Cell secretion of CGRP can promote the endothelial cell differentiation,and inhibit the differentiation to smooth muscle cells.The CGRP modification of MSCs may play a role in the regulation of angiogenesis.

AIM:Toinvestigatetheeffectsofcalcitoningene-relatedpeptide(CGRP)onmyocardinexpres-sion and phenotypic switch in vascular smooth muscle cells ( VSMCs) .METHODS:VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ( AngⅡ) , AngⅡ+CGRP or AngⅡ+CGRP +CGRP8-37 .The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot .RESULTS:The expres-sion of myocardin in cultured VSMCs showed downregulation along with time expansion .The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05).However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05).Furthermore, at 48 h in cul-tured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group .After treatment with CGRP, the levels of myocardin andα-SMA become higher ( P <0.05 ) but OPN was lower ( P <0.05 ) in CGRP group than those in AngⅡ group. CGRP8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP 8-37 group compared with CGRP group .CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein , which may be accomplished by the combination of CGRP and its receptor .

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 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 explored the effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit⁺ cardiac stem cells apoptosis. Methods: C-kit⁺ cardiac stem cells were isolated from SD rats by the methods of enzyme digestion and magnetic bead. Cells were divided into the following experimental groups: (1) negative control mimics (NCM)group: cells were transfected with negative control miRNA-21 mimics for 48 hours; (2)mimics group: cells were transfected with miRNA-21 mimics for 48 hours; (3) NCM+ H₂O₂ group: negative control miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 μmol H₂O₂ for 2 hours; (4)mimics+ H₂O₂ group: miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 μmol H₂O₂ for 2 hours. mRNA of miRNA-21 was detected by RT-PCR. The apoptosis rate of C-kit⁺ cardiac stem cells was determined using the annexin V-FITC/PI staining assay. Western blot was employed to measure the expression of apoptosis related proteins(Caspase-3, Bax, and Bcl-2). Results: (1) Compared with the NCM group, the mRNA expression level of miRNA-21 was significantly up-regulated in mimics group, while obviously down-regulated in NCM+ H₂O₂ group(all P<0.05). Compared with the mimics group, the mRNA expression levels of miRNA-21 in mimics+ H₂O₂ group was significantly downregulated (P<0.05), but remarkably upregulated compared with the NCM+ H₂O₂ group(P<0.05). (2) Flow cytometry results indicated that the early apoptosis rates were similar between the NCM group and mimics group ((4.57±3.45)% vs. (5.13±3.21)%, P>0.05). Compared with the NCM group, the early apoptosis rates were remarkably increased ((79.07±5.75)% vs.(4.57±3.45)%, P<0.05) in NCM+ H₂O₂ group. Compared with the mimics group, the early apoptosis was significantly up-regulated in the mimics+ H₂O₂ group ((30.27±1.36)% vs.(5.13±3.21)%, P<0.05), which were further down-regulated in mimics+ H₂O₂ group compared with the NCM+ H₂O₂ group ((30.27±1.36)% vs.(79.07±5.75)%, P<0.05). (3) Western blot results showed similar protein expression of Caspase-3, Bax and Bcl-2 between NCM group and mimics group(all P>0.05). Compared with the NCM group, the Caspase-3 and Bax protein expression was significantly increased in NCM+ H₂O₂ group (all P<0.05), but the protein expression level of Bcl-2 was similar between the 2 groups(P>0.05). The Caspase-3 and Bax protein expression was markedly decreased, while Bcl-2 apparently increased in the mimics+ H₂O₂ group compared with the NCM+ H₂O₂ group(all P<0.05). Conclusion Overexpression of miRNA-21 protects the C-kit⁺ cardiac stem cells from apoptosis caused by oxidative stress through downregulating proapoptotic and upregulating the antiapoptotic proteins.

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.

OBJECTIVETo investigate the impact of calcitonin gene-related peptide (CGRP) modified bone marrow mesenchymal stem cell (MSC) on the migration of vascular smooth muscle cell (VSMC) and related mechanisms.

METHODSThe MSC and VSMC were isolated from rats and cultured, CGRP was transfected to MSC with the high expression lentivirus vector, VSMC was transfected with high expression lentivirus vector of receptor activity modifying protein 1 (RAMP1) and the silence expression lentivirus vector of RAMP1. Then MSC was co-cultured with VSMC. Experimental groups were as follows: (1) Ang II group (MSC + VSMC + Ang II); (2) MSC(CGRP+) group (MSC(CGRP+) + VSMC + Ang II); (3) MSC(CGRP+) RAMP1(-) group (MSC(CGRP+) + VSMC(RAMP1-) + Ang II); (4) MSC(CGRP+) RAMP1(+) group (MSC(CGRP+) + VSMC(RAMP1+) + Ang II); (5) RAMP1(+) group (MSC + VSMC(RAMP1+) + Ang II). Transwell assay was applied to detect the migration of smooth muscle cells, Western blot was applied to detect the protein expression of cells in various groups.

RESULTSVSMC migration number was significantly lower in MSC(CGRP+) group compared with Ang II group (50.8 ± 2.6 vs. 71.4 ± 2.3, P < 0.05), but higher than in MSC(CGRP+) RAMP1(+) group (50.8 ± 2.6 vs. 30.4 ± 3.0, P < 0.05). When RAMP1 expression reduced in VSMC, compared with MSC(CGRP+) RAMP1(+) group, VSMC migration increased in the MSC(CGRP+) RAMP1(-) group compared to MSC(CGRP+)RAMP1(+) (69.0 ± 5.6 vs. 30.4 ± 3.0, P < 0.05) and was similar to Ang II group (69.0 ± 5.6 vs. 71.4 ± 2.3, P > 0.05) and RAMP1(+) group (71.6 ± 3.4). According to the result of Western blot, P-P65 protein expression in MSC(CGRP+) group was lower than that in Ang II group (0.475 ± 0.022 vs.0.642 ± 0.035, P < 0.05). P-P65 protein expression in MSC(CGRP+)RAMP1(-) group was higher than that in MSC(CGRP+) RAMP1(+) group (0.670 ± 0.030 vs. 0.373 ± 0.041, P < 0.05), and there was no difference between MSC(CGRP+)RAMP1(-) group and Ang II group (P > 0.05). P-P65 protein expression was similar between RAMP1(+) group (0.643 ± 0.039) and Ang II group (P > 0.05).

CONCLUSIONSCGRP inhibits VSMC migration through RAMP1. NF-κB and RAMP1 play crucial role in the inhibiting effects of CGRP on VSMC migration. Thus, RAMP1-CGRP signaling inhibits VSMC migration through NF-κB signal pathways.

Animals ; Bone Marrow Cells ; Calcitonin Gene-Related Peptide ; Cell Movement ; Coculture Techniques ; Hematopoietic Stem Cells ; In Vitro Techniques ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; NF-kappa B ; Rats ; Receptor Activity-Modifying Protein 1 ; Signal Transduction ; Transfection