| BackgroundPremature ovarian failure(POF) is the syndrome that female before the age of 40, normal or delayed puberty menstruation, with normal female secondary sexual characteristics, apprear persistent amenorrhea and genital atrophy, aceompanied by follicle stimulating hormone(FSH) and luteinizing honnone(LH) increase, while estrogen reduces.In the primary amenorrhea POF occupies 10% to 28%, in the secondary amenorrhea,4%-18%.It is reported that the incidence rate of POF is 1%-3.8% in China, while 1% in foreign.And in nearly 10 years, the incidence of premature ovarian failure in women of childbearing age have increased year by year and have the trend to younger age.POF is harmful to women's health, leading to a series of reproductive endcrinologic and health problems.POF needs more and more attention when its exact cause is unelear, and is difficult to treat.IPC(Intraperitoneal chemotherapy) is a regional chemotherapy that can cure cancer in peritoneum through infusing anti-tumor drug into belly cavity, according to some cancer addict implantation metastasis.This method has been applied in treating ovary cancer about 40 years. CDDP is a metal complexes, its mechanism of action in anti-tumor is crossbinding with DNA, destroy structure and function of DNA, restraint mitosis of cells.CDDP is a cell cycle-nonspecific drug;cell cyclic non-specific agent, which is applied in many diseases. Its molecular weight is heavy(300), dissolubility in water is little, but much more in ascites,and stimulus is just little to peritoneum, so CDDP become a wide-applied drug in treating ovary cancer.Mesenchymal stem cell (MSCs) is an attractive novel candidate in the present study whieh participates in nearly all of human diseases including hemopoietic stem cell(HSC) transplantation, acute graft-versus-host disease, tissue injury and recovery, inflammation and myocardial infarction. Mesenchymal stem cells (MSCs) are multiopotential progenitor cells and have been used for regenerative medicine.PKH26 is a marker with high performance in fatty series, there are fluorescein or radioactive isotope elements on the top of the marker.When this marker integrate with the double lipoids in cell serous membrane, it can label cells.Once the marker integrate with the plasmalemma, it'is difficult to separate from each other. The provocation peak of PKH26 is 488mm, and emission is 503nm.PKH26 can label neuron, neuroglial cell, bacterial, yeast, lymph, monocyte, red blood cell, endotheliocyte and cell from tumor or normal tissue. PKH26 has the advantage that a halving of cellular fluorescence is observed following each cell division, the reduction in fluorescence being readily quantified by flow cytometry.The dye has been particularly effective in monitoring the in vivo homing and proliferation of haemopoietic stem cell.PKH26 shows little or no toxicity,except for some phototoxicity following prolonged exposure of PKH26-labelled cells to excitation light.So, it'is suitable for PKH26 to study and research cell differentiation, location and transport in organism.This topic is to explore the intraperitoneal injection dose of CDDP for establish POF model in rats;And then,MSCs labeled with PKH26 are injected into progeria ovaries, and we can observe MSCs impact of ovarian function in rats after chemotherapy, in order to determine whether it had a certain role in the improvement of ovarian function after chemotherapy.Chapter 1 Establishment of rat model of CDDP-induced premature ovarian failureObjective:To find an appropriate dose of Cisplatin(CDDP) that will cause ovarian damage of female rats in vivo.Method:56 adult female Wistar rats were divided into 4 group evenly,separately for control group, low-dose group, medium-dose group and high-dose group.Rats in low-dose group, medium-dose group and high-dose group were treated with cisplatin (CDDP) with different doses(3 mg-kg-1,4.5 mg-kg-1,6.0 mg·kg-1) by intraperitoneal injection in the first and eighth day,and rats in control group were injectd by 1 ml 5% GS.Activity,eating,body weight, estrous cyclicity were observed, blood serum were collected to measure gonadal hormones(estrogen and follicle-stimulating hormone) at different times (1st,8th,12th,30th,45th,60th and 75th day), and 2 rats in every group were executed for ovaries in 8th,12th,30th, 45th,60th and 75thday.Ovaries were made into pathological section, observed their histopathology, and number of follicles. After injection 45th day,2 rats remaining in every group mated with male rats, oberving their reproductive capability.Results:(1) Before injection, weight in 4 groups was not statistically significant (F=2.013, P=0.124).After injection, weight of rats in each CDDP group declined in different degrees. Appetite was in decline.Weight in medium-dose and high-dose group dropped apparently, there were significant differences between these groups (F= 28.453,102.364 and 130.306, P=0.000).(2)Estrogen cycle in the control group still rules; Low-doses group, although had complete estrogen cycle, but extended to 8 to 9 days; In high-dose group, estrogen cycle disorders, emerges estrogen interphase in a long time, an occasional metestrus. After injection 30 days, estrogen cycle in low-dose group restore normal; After injection 45 days the medium dose reappeared estrogen cycle, but the extension still did not return to former level until two months after the treatment for drug;The high-dosage groups had been estrogen cycle disorders, performance in estrogen interphase primarily.(3)Before injection, FSH, E2 level in each drug was not statistically significant (F were 0.151 and 0.433 respectively, P were0.928 and 0.730 respectively). High-dose group successively appeared in high FSH and low E2 level after the treatment, the first 12 days after giving medicine, FSH were 4.40±0.85U·L-1,4.91±0.93U·L-1, and E2 were 48.33±16.97 pg·ml-1,29.36±12.34 pg·ml-1 respectively, compared with the control group, there were significant differences (P< 0.05), and the high-dose group of high FSH level after the treatment lasted until the 75th days, compared with the experimental group still statistically significant (P=0.004). The first 12 days after the treatment total numbers of follicles in each CDDP group were less than the control experiment, and had significant differences between the groups (F=92.523, P=0.000), among them, follicles count in medium and high dose group compared with the control group had significant difference (P=0.000). The rest of Wistar rats in groups mated with male, the control, low dose group and medium dose group delivered 26, 25 and 9 baby rats in close cage first 23.5 days,28.0 days.39.0 days, and only high-dosage groups rats were still not pregnancy after 2 months.Conclusion:6mg/kg dose of cisplatin i.p. in the first and eighth day can lead to obvious damage to the function of ovary and fertility.The way of make animal model of chemotherapy-induce premature ovarian failure has high success rate, short-cycle modeling and reliable result.lt can serve as animal models for basic research.Chapter 2 Mesenchymal Stem Cells Isolated From Rat by Whole Bone Marrow Adherent Culture in Vitro and PKH26 LabelingObjective:To explore the method of isolation and culturing of rat bone marrow mesenchymal stem cells (MSCs) with the method of whole bone marrow adherent culture, and a method of labelling rat MSCs with PKH26 in vitro.Methods:MSCs were isolated and cultivated from the bone marrow of rat by whole bone marrow adherent culture. Further purification was achieved by expansion at serial passages. The passage 3 MSCs were cultured and labeled with PKH26 according to the manufacturer's instruction. The growth, fluorescence intensity and serial subcuhivation of labeled MSCs were analyzed with fluorescence microscope. The proliferation ability of these labeled cells were tested by MTT.Results:Many cell formations with bad refraction can be observed after three days whole bone marrow adherent culture.All the marrow train separation Cultured cells by more than JiLa sample distribution cloning group, refraction sex differences. The first four days the number of cells significantly increased, form of cells were rich. The stick wall area of cells reach 80%-90%, cells closely spreaded like a spiral shape or shoals shape. Spread to the 3rd generation cell with high purity already was available. Flow cytometric analysis detected 3rd generation of MSCs CD29, CD44, CD90.They were tending to high expression. After labeling, dye uniform distributed in the cell membrane, growth form of the cells unchanged. Red fluorescent weakened gradually when amplified to 3rd generation,showing granular points of light distribution. Labeled cells fluorescence intensity decreased after frequent passage. Determined by MTT method To detect cell proliferation capacity of PKH26 labeled MSCs and cells without markers,we found there was no significant statistically. Conclusion:MSCs can be successfully cultivated by whole bone marrow adherence method which is a convenient method. Labeling the MSCs with PKH26 is an effective and practical method, which can be used as an important tool in the study on transplantation of MSCs.Chapter 3 The effect of MSCs on CDDP-induced ovarian damage in ratsObjective:To study the therapeutic effect of MSCs against CDDP induced ovarian damage on rats in vivo. PKH26-labelled cells are injected to each ovary.Observing structural changes and functional restoration of ovarian tissue, exploring MSCs to improve ovarian function after chemotherapy. Researching to slow down clinical physiology of aging, treatment by chemotherapy-induced infertility or premature menopause on women.Methods:88 female Wistar rats were randomly divided into four groups:â‘ treatment group(22),Experimental control group(22)and blank control group (22);â‘¡blank group (22):without any drug treatment The three front groups were used the same methods and the same dose to establish ovarian failure model by chemotherapy, specific methods are as follows:6mg/kg dose of cisplatin i.p. in the first and eighth day. The first 13-day experiment, three groups that were open lines to inject double-ovarian. Experimental control group was received normol saline (NS) 0.1ml; blank control group:without any drug treatment; treatment group:received PKH26-labeled MSCs 0.1ml 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, and respectively killed five rats fifty days,thirty days,forty-fivedays and sixty days later, collected the 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 change of fluorescence intensity of coal maceral. After MSCs transplantion 30 days, two rats of each group were taken to mate with male rats in order to observe the difference of their reproductive activity..Results:1.Blank 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 after administration.The 30th days after cells transplantation, 23.53% rats in treatment group resume to intact estrous cycle, about 6-8 days; but experimental control group and blank control group of estrous cycle has yet to resume at the end of the experiment.2. During the experiment, sex hormone level of blank group did not happen obvious change. Before MSCs transplantation, FSH and E2 concentration ofâ‘ â‘¡â‘¢groups of rats compared with each other, there were no statistically significant differences, respectively (F=0.195 and 0.052, P=0.826 and 0.950), but on the whole, mean differences of four groups are statistically significant (F=5.309 and 7.207, P=0.010 and 0.003).15 days after MSCs transplantion, treatment group FSH concentration before transplantation already from the (5.11±1.22)U·L-1 fell to (4.84±0.49) U·L-1, below experimental control group and blank control group level (5.60±1.60 and 5.73±1.41)U·L-1, but still higher than the blank group level (3.03±0.72) U·L-1; For the same time, treatment group E2 concentration (55.12±9.09)pg·ml-1, higher than level before transplantation (28.98±16.10) pg·ml-1, and also higher than experimental control group and blank control group level (30.79±6.02 and 27.23±9.69) pg·ml-1, but still lower than the same period (65.76±21.06) pg·ml-1; The other three group compared with treatment group, there was a statistically significant difference(F=5.803 and 11.390, P=0.002 and 0.007 respectively). The first 30 days after transplantation MSCs, treatment group for FSH concentration (4.33±1.09) U·L-1 declined 15 days after transplantion, more below than experimental control group and blank control group level (5.98±1.09and6.33±1.51) U·L-1, but still higher than the blank group level (3.12±1.02) U·L-1;The experimental E2 level rises (56.52±8.70) pg·ml-1, still lower than the same blank group (64.14±26.61) pg·ml-1, meanwhile, experimental control group and blank control group were still wandering in(23.21±9.61å'Œ26.21±13.62) pg·ml-1. The other three groups were compared with treatment group were statistically significant difference (F=7.893 and 8.179, P=0.002), respectively. The first 45 days after MSCs transplantation, experimental FSH concentration for (3.95±1.13) U·L-1, still higher than the blank group (2.77±1.18) U·L-1, more below than experimental control group and blank control group level (6.09±1.27and6.67±0.92) U·L-1; The E2 level in treatment group more increases (66.51±11.23) pg·ml-1, still lower than the same blank group (67.25±22.07) pg·ml-1, meanwhile, experimental control group and blank control group were still wandering in (28.59±6.80 and 25.25±10.67) pg·ml-1. The other three groups compared with treatment group were statistically significant difference, respectively (F=12.951 and 13.433, P are 0.000).60 days after MSCs transplantation, experimental FSH concentration has fallen to (3.59±0.99) U·L-1 although still higher than the same blank group concentration (3.13±1.24) U·L-1 and dosing made before this mould (2.97±1.23) U·L-1, but the difference was not statistically significant (P=0.458 and 0.41, respectively); Experimental E2 concentration (64.57±14.52) pg·ml-1 also is below the blank group E2 (69.25±15.06) pg·ml-1 and dosing made before this mould (72.24±23.86) pg·ml-1. but also no statistically significant (P=0.566 and 0.527), respectively.3.The 15th days after cells transplantation, the number of follicles of experimental group compare with experimental control group, blank control group, the differences were statistically significant(P<0.05). The 60th days after cells transplantation, the differences remained. 4.15 days after MSCs transplantation, the ovarian frozen section was observed though fluorescence microscopy.MSCs mainly focus on the needle into the part when transplanting syringe, mainly for ovarian stromal department, some cells scattered around the marked cells clump.30 days after MSCs transplantation, red remain visible fluorescence, and fluorescence is spread a bit, but fluorescence intensity of uniform has been downgraded. After transplantation MSCs 45,60 days, fluorescence has a gradual weakening trend. Four points in time there were no oocytes show red fluorescent.5.At last, blank group and treatment group could give birth to baby mice, none of experimental control group or blank control group pregnancy.Conclusion:MSCs can improve part of ovarian function in rats:1. Cisplatin can cause female rats E2 reducing, after MSCs transplantation in ovaries. E2 have rebounded, possibly MSCs suppresses the granulosa cells which can secrete E2 excessively apoptosis, delaying the follicle atresia. thereby reducing the closure of cisplatin in body ovarian damage; 2. MSCs may repair an ovarian stromal fibrosis. and thus, prevent ovaries to chemotherapy injury, make the reproductive capacity from damage; 3.The detailed mechanism of MSCs treatment chemotherapy traumatic ovaries, still need to further research. |
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