Experimental Research Of Bcl-2 Gene Expression And Impact Of Ovarian Function Caused By Chemotherapy By Means Of Lentivirus Vector Mediation In Rats

BackgroundPremature ovarian failure(POF),also known as post-menopausal syndrome,is mainly expressed function cessation of activities in ovarian.Normal women in the 40-year-old ovaries gradually stop their activities.In recent years the incidence of POF increased year by year,patients added with exogenous hormone clinically can improve symptoms,prevent osteoporosis and improve the quality of life.But it has side effects,such as long-term medication and poor compliance,and can not solve anovulatory infertility problem caused by POF.Therefore it is necessary to study it depty.POF is caused by a variety of reasons,which occurs in many women under 40 years of age.Clinical manifestations is characterized as secondary or primary amenorrhea,a reduction in the level of serum estradiol(E₂＜25 pg/mL) and increased gonadotropin level(FSH＞40U/L),and have different degrees of a series of low-estrogen symptoms,such as:hot flashes,excessive sweating,facial flushing,low sexual desire.Tsujimoto cloned a kind of anti-apoptosis gene from follicular lymphocytes tumor-related t(14;18) chromosomal translocation breakpoint in 1984,which was named as Bcl-2(B-cell lymphoma 2) gene.Bcl-2 gene encodes a protein of 25-26KD, its 21 hydrophobic of C terminal amino acid composes an extensive chain structure. This chain can be inserted in the cell membrane structure,the structural characteristic is relevant to Bcl-2 regulation of apoptosis.Bcl-2 has confirmed in the mitochondrial outer membrane,nuclear membrane and endoplasmic reticulum.According to the structure research of Bcl-2 family members protein,and it has found common ground is that exist three structural motifs in all the protein chain,which known as BH1,BH2 and BH3 domain respectively.Lentivirus vector is a gene therapy vector,which is based on HIV-â… (human immunodeficiency virus typeâ… ).It is relatively site-specific integrated into chromosome specific location,and high-level expressed in the split and non-dividing cells.Studies have shown that HIV-â… as a basic construct of these lentiviral vector,which can infect non-dividing cells,integrate the target gene into the target cell genome.Because it has long-term expression and small immune response,so it is suitable for gene therapy in vivo.It is guaranteed that HIV-â… vector was safe,because there is not copied force HIV in all vitro and out vivo experiments.Green fluorescent protein(GFP) is a light-emitting protein which was first isolated and purified from the hydra and jellyfish Aequorea Victoria animals by Shimomura and Johnson.This protein is encoded by the GFP gene,can produce green or yellow-green fluorescence under the blue light excitation,and can be detected by fluorescence activated cell sorting device and fluorescence microscopy.GFP does not need to add any cofactors to detect,detectes facilitately and high sensitivity;maintains fluorescence stability,long fluorescence and doesn't interfere with normal ovarian structure and function.Enhanced green fluorescent protein(EGFP) is a GFP mutant; 64 points Phe→Leu,65 sites of Ser→Thr,which emittes GFP fluorescence intensity 6 times higher than major.Therefore,as a reporter gene is more suitable than GFP in studying gene expression,regulating,cell differentiation,positioning and transfering protein in organisms.In recent years,researchers have detected B-cell lymphoma(Bcl) -2 gene in ovarian tissue,which can induce granulosa cell to secrete of Bcl-2 protein,regulate granulosa cells and oocyte cells in the proliferation and differentiation by autocrine / paracrine,and it is necessary for the continued development follicles of regulatory proteins.Therefore,this topic was to explore lentivirus-mediated Bcl-2 gene expression in rats and inpact of ovarian function after chemotherapy,in order to determine whether it had a certain role in the improvement of ovarian function after chemotherapy.ObjectiveIn this study,exploring lentivirus-mediated Bcl-2 gene expression in rats and set -ting up animal models of ovarian failure by intraperitoneal injection of cyclophosphamide(CTX).Bcl-2 gene carrying enhanced green fluorescent protein (EGFP) was mediated by lentiviral vector,injected to each ovarian.Detecting apoptosis,Observing structural changes and functional restoration of ovarian tissue,exploring the Bcl-2 gene to improve ovarian function after chemotherapy. Researching to slow down clinical physiology of aging,treatment by chemotherapy-induced infertility or premature menopause on women.Materials And Methods1.SPF level female Wistar rats,2-3 mouths old,weight 180-200g.Vaginal smear were obtained everyday at 9:00 am to assess ovulatory status.Estrous cycle were observed under microscope rats,which had a normal estrous cycle entered the experiment. 2.Pre-experiment:explore the minimum effective dose of empty lentiviral vector transfected normal ovarian tissue in rats:12 female Wistar rats were randomly divided into 3 groups,4 in each group are chloral hydrate by 3ml / Kg by intraperitoneal injection of anesthesia,open the ventral abdominal line to expose the ovariectomy.Three groups of rats were injected ovariectomy with the substance of enhanced green fluorescent protein(EGFP) of the empty-HIV-â… respectively,but different doses.Group 1:1μl;Group 2:5μl Group 3:10μl.After injecting,observe daily feeding,toilet,activities,body hair,weight,and the situation of the estrous cycle.7,14,21,28 days after injection rats were killed(one for each time point of low-dose group n = 1,middle dose group n = 1,high-dose group n = 1),executed ovariectomy respectively,weighted out.The expression of EGFP was given by fluorescene microscope.At the same time,ovarian changes in organizational structure by HE staining.3.The effects of ovarian function after chemotherapy by lentivirus-mediated Bcl-2 gene:60 female Wistar rats were randomly divided into four groups:â‘ blank control group:without any drug treatment;â‘¡Experimental control group(15),no-load control group(15) and treatment group(15).The three groups were used the same methods and the same dose to establish ovarian failure model by chemotherapy, specific methods are as follows:8mg/(kg·d) by i.p.after a 50mg/kg loading dose for total of 14d.The first 16-day experiment,three groups that were open lines to inject double-ovarian.Experimental control group was received normol saline(NS) 5μl; no-load control group:received CTX+HIV-â… 5μl;treatment group:received Bcl-2-HIV-â… 5μl respectively.From the start of the experiment to the end,rat estrous cycle was monitored by vaginal smear method,E₂,FSH were detected every 15 days(equivalent to three normal rat estrous cycle time),and respectively killed half of the rats fifty days and thirty days later,collected the uterus and ovaries,respectively, all specimens were paraffin-embedded and frozen line sections with hematoxylin-eosin(Hematoxylin-Yihong,HE) staining to compare the weight of the ovary and the numbers of the follicle.Cryo-sectioning fluorescence microscopy to observe the expression of enhance green fluorescent protein.Using TUNEL assay to detect ovarian granulosa cell apoptosis and Western blot to detect anti-apoptotic protein Bcl-2 expression.Results1,Determine the minimum effective dose of empty lentiviral vector transfection of rat ovarian tissue(pre-experimental)Bilateral ovarian injection of different doses of empty lentiviral vector,the three groups of rats with normal eating,toilet,weight gain,activity-sensitive,light moisturizing hair and a normal estrous cycle.7,14,21,28 days after injection rats were killed respectively,then weighted out.There was no statistically significant among three groups of ovarian weight(P＞0.05).Ovarian lines after frozen section was observed expression of EGFP under fluorescence microscope.Low-dose group was only saw part of ovarian tissue to appear weak EGFP expression in the first 7,14,21,28-day,infection efficiency was 40%;the first 7,14,21 of EGFP expression gradually increased in medium-dose group,and 28 days had stable expression, infected efficiency was more than 90%;High dose group from the first 7 days to 28 days was saw a strong expression of green fluorescence,but the strong background color was easy to influence the accuracy of observation.Three groups of rats were on the Paraffin-embedded,continuous slice,hematoxylin - Yihong(HE) staining and pathology,lentiviral vector injection ovarians were no obvious degeneration and necrosis pathological abnormalities under the microscope..Low-dose group was low transfection efficiency,medium and high dose group of the empty lentiviral vector can be effectively transfected into the rat ovarian tissue,there was no significant general situation and changes in ovarian weight among three groups of rats.As a result of small ovarian tissue,such as injected too much liquid may have a certain impact on ovarian function.Therefore,the lowest effective dose was the 5μl in follow-up experiment(medium-dose group).2,E₂,FSH and estrous cycle changes in each different intervention methods by lentivirus-mediated Bcl-2 gene on ovarian failure after chemotherapy(1)Blank control group of rats during the entire experiment vaginal smear was normal estrous cycle changes;Vaginal smears showed that the other groups' rats was sustained non-ovulation in the 3～5 days after administration.After stopping 15 days treatment group resume to normal estrous cycle;but experimental control group and no-load control group of estrous cycle has yet to resume at the end of the experiment.(2)Theere were no significant differences among experimental group, experiment control group and no-load control group rats in the beginning of the experiment in FSH,E₂ concentrations,but compared with the first 15 days of hormone concentrations,there were significantly different(P＜0.05).After stopping CTX 15 days,treatment group of serum FSH concentrations was(3.74±0.10) mIU / ml,higher than the corresponding blank control group(3.33±0.08) mIU/ml,lower than the experimental control group(4.98±0.11) mIU /ml and the no-load control group(5.10±0.10) mIU / ml,also lower than the stopping day in this group(4.67±0.09) mIU/ml;E₂ concentrations beginning from the first rise,as (39.54±1.99) pg / ml,lower than the same period in the blank control group(51.99±2.11) pg/ml,higher than the experimental control group(30.37±1.50) pg / ml and the no-load control group(29.44±1.16) pg / ml respectively,also higher than the stopping day of the itself level(31.10±0.91) pg/ml.Compare with FSH and E₂ in the same period,there was statistically significant differences between experimental group and other three groups(P＜0.05).After stopping CTX 30 days,treatment group of serum FSH was(3.66±0.06)mIU/ml,still higher than the corresponding blank control group(3.36±0.12) mIU/ml,lower than the experimental control group and the no-load control group of FSH,(5.65±0.08) mIU / ml and(5.66±0.08) mIU / ml respectively,the differences were statistically significant(P＜0.05);Experimental group,serum E₂ concentration significantly increased,as(43.79±3.17) pg / ml,also lower than the same period in the blank control group(51.29±1.94) pg/ml,and the experimental control group and the no-load control group continued to decrease,respectively(29.41±0.62) pg / ml and(29.07±1.20) pg / ml.Compare with FSH and E₂ in the same period,there was statistically significant differences between experimental group and other three groups(P＜0.05).Compare with E₂ and FSH in the same period,there wasn't statistically significant differences between experimental control group and no-load control group(P＞0.05).3,Ovarian weight changes in each group by lentivirus-mediated Bcl-2 gene on ovarian failure after chemotherapyAfter stopping CTX 15 days,treatment group of ovarian weight was(46.00±0.87) mg,lower than the same period in the blank control group(51.38±1.54)mg, higher than experimental control group was(40.31±1.34) mg and no-load control group was(41.50±1.34) mg respectively.There were statistically significant differences between experimental group and other 3 groups(P＜0.05).There weren't statistically significant differences between experimental control group and no-load control group(P＞0.05).After stopping CTX 30 days,treatment group of ovarian weight was(49.99±0.73) mg,still lower than the blank control group(51.85±1.18)mg,experimental control group and no-load control group of ovarian weight steadily reduced,as(32.72±1.26) mg and(33.37±1.84)mg.There were statistically significant differences between experimental group and other 3 groups(P＜0.05).There weren't statistically significant differences between experimental control group and no-load control group (P＞0.05).4,Numbers of follicle changes in each group by lentivirus-mediated Bcl-2 gene on ovarian failure after chemotherapyAfter stopping CTX 15 days of the experimental phases,the number of follicles of experimental group compare with experimental control group,no-load control group,the differences were statistically significant(P＜0.05).Treatment group proportion of primordial follicles gradually reduced,mature follicles gradually increased.But the total number of follicles and each period number of follicles continued to decrease in experimental control group and no-load control group,one of the most obvious is mature follicle.After stopping CTX 30 days,follicular phases of the experimental group were higher than the number of experimental control group and no-load control group,the period of follicle quantity compared with the other three groups,the differences were statistically significant(P＜0.05).5,Toxicity test:Sacrificed ovarian and uterine from no-load control group and experimental group of Wistar rats,two groups of rats could be seen strong fluorescence,but the uterus couldn't be seen fluorescence.Lentiviral vector injection ovarians were no obvious degeneration and necrosis pathological abnormalities under the microscope.6,TUNEL assay apoptosis index:after stopping CTX 30 days of the experiment,A I of treatment group was(6.44±0.78)%.There was significant difference among blank control group(2.58±0.26)%,experiment control group(18.53±0.84)%,no-load control group(18.94±0.43)%(P＜0.05). 7,Western blot detect Bcl-2 protein expression:after stopping CTX 30 days of the experiment,Bcl-2 gene were detectable in the treatment group and were significant higher than in the other two groups(P＜0.05 ).The quantitative of experimental group rats of Bcl-2 protein expression was 1.75 timer than experiment control group,1.47 timer than no-load control group.Conclusions1,The study found that lentiviral vector can be successful transfected into rat ovarian tissue,the body weight of rats and the organizational structure of the ovary had no significant changes,in the near organs the uterus without be found fluorescence expression,the virus is confined to the local situation.It is guaranteed that HIV-â… vector was safe,it lays the foundation for clinical trials in the future.2,entivirus-mediated Bcl-2 gene successful transfected into rat ovarian tissue, it can inhibit rat ovarian granulosa cell apoptosis,due to the promotion of antiapoptotic Bcl-2 protein secretion.3,lentivirus-mediated Bcl-2 gene can improve part of ovarian function in rats, the possible mechanisms the level of Bcl-2 gene was increased,then inhibited rat ovarian granulosa cell apoptosis,promoted granulosa cell to secrete estradiol.4,Because of the limited experimental conditions,failed to mate after stopdrug, the reproductive function in rats whether or not to resume,pending further experiments to prove beyond.