Expression Of A Subfamily Member Of Transient Receptor Potential Gene (TRPV4) In Rat's Testis

BackgroundIn the cell environment,the calcium ion is the key informational molecule and the only way to trigger cell proliferation while the intracellular calcium ion concentration in cytoplasm is regulated by the cell membrane calcium channel.The latest research on the cell membrane calcium channel shows that,it's the only way for calcium influx induced by cell membrane activated calcium channel to trigger cell proliferation;non-voltage-dependent calcium channel,Transient Receptor Potential (TRP),has a function to regulate cell survival,growth,death and so on.At many TRP-mediated functions and disease,it at the role of cell proliferation is particularly prominent,which in many tissues and ceils,have been supported by experimental results.For example,the pulmonary artery smooth muscle cells of TRPC1 and TRPC6 expression was significantly higher,and their by-mediated calcium influx stimulate smooth muscle cell proliferation,which eventually led to the occurrence of pulmonary hypertension;source of vascular endothelial growth factor (VEGF) by activating endothelial cells TRP channels,causing calcium influx, stimulating vascular endothelial cell proliferation can lead to tumor angiogenesis; TRPM1 expression and the malignant melanoma is closely related to the degree; These results indicate that,TRP in the regulation of cell proliferation has a role that can not be ignored.At present,many studies have found that,TRP gene family at a number of subtypes have on the expression of the testis,in part or high expression.However, research in this regard is still very small,it is necessary to study.At the above-mentioned study on the testis epididymis are limited to the question whether the expression,since the TRP family in the testis on the expression of such a rich place in the testicular function on it becomes a concern,and on current international research is still There is no relevant research reports.On the current international research shows that:TRPV4 substantial expression in renal distal convoluted tubule of the epithelial tissue,but also a small amount of expression in heart,liver,lung,spleen,testis,and many other organizations.Although functional studies have made considerable achievements,such as:TRPV4 in airway smooth muscle cells may relate to airway hyperresponsiveness,such as asthma; TRPV4 at the expression of Corti may indicate sensorineural hearing damage; TRPV4 with ewes caused by temperature change on the micturition reflex.However, in TRPV4 on testicular function has not yet reported.To study its relationship with the testis will have important practical significance and broad prospects for development.Therefore,the study of TRPV4 and testis,as well as on the distribution function is of great significance.To this end,we experiment on animals through TRPV4 channels and the relationship between testicular tissue for a preliminary study.1 PurposeTo study the expression of TRPV4,a subfamily member of TRP gene in rat's testis.To Explore the TRP gene family in testis on the expression for the testicular tissue on TRPV4 function of laying the foundation.2 Method2.1 RT-PCR detection of TRPV4 in the rat testis tissue mRNA expression 2.1.1 TRPV4 gene primer designTRPV4 landing rats genebank,using Primer Premer5.0 software to design primers.2.1.2 Total RNA Extraction and cDNA SynthesisRats by intraperitoneal injection of phenobarbital death,testieular tissues collected and immediately stored into liquid nitrogen.100mg from testieular tissue,in accordance with the common method for molecular cloning TRIzol extracted total cellular RNA;then at 37℃with DNaseI digestion 30min,to remove possible DNA contamination.Nucleic acid/protein quantitative determination of its A260 spectrophotometer and A280.For A260/A280 greater than 18 but less than 20 of the RNA samples,check lug total RNA,in accordance with the instructions synthesis reagents cDNA.2.1.3 RT-PCRThe first chain as a template,amplified by the following conditions:94℃10min, 94℃45s,55℃45s,72℃1min,30cycles,then 94℃1min,60℃1min,72℃10min.PCR products line 0.7%agarose gel eleetrophoresis.Gel imaging Analyzer.2.2 Immunohistochemistry Detect TRPV4 protein in rat testis tissues2.2.1 TRPV4 buy monoclonal antibody and SABC immunohistoehemistry kit TRPV4 monoelonal antibodies.2.2.2 Specimen acquisition and handling:Rats by intraperitoneal injection of phenobarbital death,collected testicular tissue into formalin solution fixed,by the ladder dehydration,xylene and the Baptist transparent wax,the paraffin-embedded tissues.2.2.3 ImmunohistochemistryUsing SABC method,all tissue samples with thickness of 4μm serial sections 3, in accordance with the instructions reagents operation,the use of phosphate buffer solution(PBS) in place of anti-1 for negative control.To the cytoplasm showed brown particles for positive expression.2.3 Western blot detection of the third part of TRPV4 protein in rat testis tissues2.3.1 Specimen acquisition and processingRats by intraperitoneal injection of phenobarbital death,collected testicular tissue,using protein extracted protein extract,using pre-cooling of the PBS wash 2 times,go supematant,Canada on 4×SDS gel sample buffer,denatured in boiling water bath 10min.2.3.2 Western blotPreparation of conventional SDS-PAGE gel,two sets of the same sample points, 120V about 3.5h after electrophoresis,cut one of a group by conventional methods fixed,Coomassie brilliant blue R-250 dyeing and bleaching,as a judge based on Western blot.Another group of samples to cut plastic membrane,nitrocellulose membrane by tailoring the size of gel and do a good job in the beginning and end tags, filter paper on both sides of the buy 3,according to plastic film negative is placed,4℃,18V switch membrane overnight.Remove the membrane with liquid closed closed at least 2h,TBS washing liquid membrane 3 times 10min;add 1:100 diluted anti-1,4℃role 2h;TBS washing liquid membrane 3 times 10min;add 1:5000 diluted goat anti-rabbit IgG secondary antibody at room temperature,the role of 1h; TBS washing liquid membrane 3 times 10min;imaging technology to observe by reflection band.Gel Logia 200 with camera imaging system and analyzed.3 Results3.1 RT-PCR detection of TRPV4 in the rat testis tissue mRNA expressionUV instrument at the next transmission,TRPV4 the lane at the standard nucleic acid molecular weight near a specific amplification bands of approximately 237 bp fragment about the size,image analysis showed that the product of higher purity,with no more than specific interference.3.2 Immunohistochemistry Detect TRPV4 protein in rat testis tissues TRPV4 can be seen under the microscope-positive film,brown many antigen-antibody complex deposition in the testicular tissue in the more mature spermatogenic cells and some Sertoli cell nuclei;TRPV4 negative film at brown no antigen-antibody complex deposition.3.3 Western blot detection of TRPV4 protein in rat testis tissuesWith Gel Logia 200 Imaging System camera,the picture shows:In the test lane, in the 36KDa and 871KDa Department clearly shows that the positive bands,and in the negative control lane,only to see the positive in the Department 36KDa bands can be identified in rat Testicular tissue has TRPV4 protein and GAPDH protein expression,GAPDH protein as a reference.4 ConclusionTRPV4 in the testicular tissue has on the expression,the main expression in more mature spermatogenic cells and supporting cells,showing strong expression.