The Effect Of Adenovirus-Dilivered ShRAN Targeting BFGF On The Proliferation And Apoptosis Of Prostatic Stroma Cells

ObjectiveBenign prostatic hyperplasia(BPH) is a common disease in elder men, which can lead to complicated lower urinary tract symptoms and do harm to the heath of old men. With the social development and the aging tendency of population, there is a sharp increasing mobidity rate of BPH. It can be treated by medication or by surgery. The former, including a adrenergic receptor antagonists and 5a-reducase inhibitor, do not show a satisfactory effect, while the latter is traumatic though it's effective. So we should try to find other effective and safe way to treat BPH.The prostatic tissue is composed of epithelia and stroma. The stroma area is twice as large as the epithelia area in normal prostate, while in BPH, the ratio increases to 5. Moreover, BPH begins with the stroma hyperplasia. In the prostatic stroma, there accumulate a great deal of smooth muscle cells and fibroblasts.Basic fibroblast growth factor(bFGF) widely spread in the human tissues such as heart, brain, liver, kidney, and so on. As a strong mitogen, it can promote the proliferation, differentiation and migration of various kinds of cells. There are high level expressions of bFGF in some abnormal hyperplasia tissues or tumor. Inhibition of bFGF or its receptors will inhibit proliferation of many kinds of cells, promote their apoptosis. Many studies have shown that there is a high level expression of bFGF in the BPH tissue, which results in the proliferation of prostatic cells by autocrine and paracrine, and plays an important role in the development of benign prostatic hyperplasia. This study make a further survey of the relative expression level of bFGF in the epithelia and stroma of prostate.RNA interference(RNAi) is a a gene silencing phenomenon whereby specific dsRNAs trigger the degradation of homologous mRNA. PNAi can silience the target gene specifically and efficiently, which shows a perspective in the study of gene function and gene therapy. Adenovirous vector is the most widely used tool of gene transfer for its high efficiency of infection, wideness of host cells and steady effect of transfection. To construct adenovirous vector delivering special shRNA targeting bFGF can provide a effective tool for the study of many kinds of hyperplasia deseases.Using Adeno-XTM Expression Systems 2, we construct adenovirous5 vector delivering special shRNA targeting bFGF, and infect cultured prostatic stroma cells to investigate the bFGF expression and the proliferation and apoptosis of the cells.MathodDetect the bFGF expression in the epithelia and stroma of normal and hyperplasia prostatic tissue by immunohistochemical method. Microimage analysis of 5 random field of each slide, determine the average optical of the whole field, the epithelia area and stromal area.Construct adenovirous5 vector delivering special shRNA targeting bFGF, mainly 3 steps:(1) synthesize 19nt shRNA oligonucleotide targeting bFGF CDS (accession number NM002006.4800-818), anneal, connect the double strand shRNA with the digested donor vector pSHREN-DNR to form the recombinated donor vector pSHREN-DNR-sh-bFGF, verify by PCR and sequencing; (2) recombinate donor vector pSHREN-DNR-sh-bFGF with acceptor vector pLP-Adeno5-X-PRLS to form recombinate adenovector pAd5-sh-bFGF with bFGF shRNA expression box and adenovirus5 DNA(△E1/△E3) and Cm resistance gene; (3) package and amplify adenovirus partical:transfecting HEK 293 Cells with Pac I-digested pAd5-sh-bFGF to get the primary virus stock and amplify. Construct non-sense control adenovirus5 Ad5-sh-non simultaneously. Determine the tilter of the adenoviruses. Culture human prostatic stromal cells. Infect prostatic stromal cell with Ad5-sh-bFGF,Ad5-sh-Non and Ad5-EGFP (replication defective Adenovirus5 expressing EGFP) respectively with MOI=0.1,1,10 and 100. Detect proliferation with MTT. Calculate proliferation inhibition raio=(1-Aexperimental goup/Acontrol) X 100%. Detect apoptosis using FCM by AnnexinV PI double dye. Detect bFGF mRNA expression by real time PCR. Detect bFGF protein by western blot.ResultThere is a higher expression level of bFGF in BPH tissue compared with normal adult prostatic tissue with significant diference(P<0.01). bFGF expression level in stroma is higher than that in the epithelia (P<0.01).We successfully constructed Adenovirus5 vector delivering bFGF shRNA (Ad5-sh-bFGF) and nonsense control(Ad5-sh-Non).3 days after infection by Ad5-sh-bFGF, when MOI=0.1,1,10,100, the proliferation inhibition rates are 5.68%,8.57%,12.63% and14.93% respectively. When MOI=10, the proliferation inhibition rate is significant higher than MOI=0.1 or 1(P<0.01). There is no significant difference between MOI=10 ane 100(P>0.05). When MOI=10, the infection efficient is close to 100%.3 days after infecting cultured prostatic stromal cells with Ad5-sh-bFGF, Ad5-sh-Non and Ad5-EGFP(MOI=10), proliferation inhibition rate is 12.63%,4.93% and 4.08% respectively, there are significant differences between Ad5-sh-bFGF group and the latter two groups (P＜0.01), while there is no significant difference between Ad5-sh-Non and Ad5-EGFP group(P>0.05).3 days after infection by Ad5-sh-bFGF(MOI=10), the apoptosis rate of cultured prostatic stromal cells is 10.51+1.66%, obviously lower than those of Ad5-sh-Non, Ad5-EGFP and blank group (P＜0.01).Using real time PCR to detect the bFGF mRNA expression of cultured prostatic stroma cells 3 days after infected by Ad5-sh-bFGF(MOI=10), the relative ratio (bFGF/β-actin) is 0.431±0.097, significantly lower than those of Ad5-sh-Non, Ad5-EGFP and blank group (P＜0.01).Using western blot to detect the bFGF protein expression of cultured prostatic stroma cells 3 days after infected by Ad5-sh-bFGF(MOI=10), the relative ratio (bFGF/β-actin) is 0.260±0.062, significantly lower than those of Ad5-sh-Non, Ad5-EGFP and blank group (P＜0.01). There is no significant difference among the latter 3 groups(P>0.05).ConclusionCompared with normal prostate tissue of adults, there is a higher expression level of bFGF in BPH tissue. bFGF expression level in stroma is much higher than that in the epithelia.We successfully constructed Adenovirus5 vector delivering bFGF shRNA (Ad5-sh-bFGF) and when infect cultured prostatic stromal cells, it can inhibit the proliferation, and promote the apoptosis of the cells, which is due to the specific inhibition of bFGF expression.