鸡输卵管上皮细胞体外分离培养的研究

鸡输卵管上皮细胞是卵清蛋白的主要分泌细胞,是研究输卵管特异表达蛋白调控的重要工具。在以往的研究中,多采用普通DMEM培养液对鸡输卵管上皮细胞进行分离与培养,容易造成其自身特性在体外培养过程中的改变。本研究我们优化了细胞分离方法,发现从输卵管漏斗部组织分离的输卵管上皮细胞增殖较快;用鸡输卵管上皮细胞培养基相比DMEM更适合促进细胞生长;与胰酶相比,用Accutase消化酶进行细胞传代,有利于输卵管上皮细胞特性维持。对所获得的输卵管上皮细胞鉴定发现,己烯雌酚能促进卵清蛋白的表达,说明分离培养的细胞保持了鸡输卵管上皮细胞特性。本研究建立的方法为输卵管特异表达蛋白调控以及家禽生物反应器的研究奠定了基础。     

Effect of progesterone administration on the distribution of oviductal carbohydrates in <Emphasis Type="Italic">Rana tigrina</Emphasis>

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed. This research was supported by Thailand Research Funds (to W.W.), a Research Initiate Grant from Kasetsart University (to A.T.), and Mahidol University.     

Estrogen Receptor Is Not Primarily Responsible for Altered Responsiveness of Ovalbumin mRNA Induction in the Oviduct From Genetically Selected High- and Low-Albumen Chicken Lines

The role of estrogen receptor on ovalbumin mRNA induction by steroid hormones was investigated in primary cultures of oviduct cells from estrogen-stimulated immature chicks of genetically selected high- and low-albumen egg laying lines (H- and L-lines). In experiment 1,the extent of ovalbumin mRNA induction and changes in estrogen and progesterone receptors were compared between the oviduct cells from H- and L-lines with or without steroid hormones in the culture medium. In experiment 2, the effect of estrogen receptor gene transfection on the induction of ovalbumin mRNA was studied in the oviduct cells from the L-line chicks. The results showed a close correlation of the changes in ovalbumin mRNA with the numbers of nuclear and total estrogen receptors in the oviduct cells but not with the numbers of nuclear and total progesterone receptors. Estrogen receptor gene transfection induced ovalbumin mRNA to a moderate extent in the absence of the steroid hormones. To our surprise, however, estrogen receptor gene transfection apparently suppressed the ovalbumin mRNA responsiveness to estrogen to a considerable extent. It was concluded, therefore, that the extent of estrogen receptor expression might not be primarily responsible for the differences in responsiveness to steroid hormones of oviduct cells from genetically selected H- and L-line chickens.     

Effects of serial hormone treatments on egg white protein synthesis. Further evidence of translational regulation

The administration of either progesterone or estrogen to withdrawn chicks several hours after a first dose of estrogen affected ovalbumin synthesis differently than its mRNA levels [S. S. Seaver (1981) J. steroid Biochem. 14, 949-957]. This suggested that the hormones were regulating the translation of ovalbumin directly. In this paper we report that serial hormone treatments also affect the rates of synthesis of two other egg white proteins, conalbumin and ovomucoid. When progesterone was administered 4 h after estrogen, conalbumin synthesis decreased. When either progesterone or a second dose of estrogen was administered 12 h after the first dose of estrogen, conalbumin synthesis increased. Serial hormone treatments did not always affect all three proteins similarly. At later times, administering progesterone after estrogen decreased ovomucoid synthesis but did not affect conalbumin or ovalbumin synthesis. To determine if the serial hormone treatments affect egg white protein mRNA's in a similar way, changes in ovalbumin and conalbumin mRNA levels were quantified in a rabbit reticulocyte cell-free translation system and were compared to changes in ovalbumin and conalbumin synthesis as measured in chick oviduct tissue minces. When serial hormone treatments were 12 h apart, ovalbumin and conalbumin synthesis was 50-300% higher than that predicted by the changes in ovalbumin or conalbumin mRNA levels. This is further evidence that translation of both conalbumin mRNA and ovalbumin mRNA is directly regulated by steroid hormones.     

Expression of the avian c-erb B (EGF receptor) protooncogene during estrogen-promoted oviduct growth

Expression of cellular erb B protooncogene messenger RNAs has been analyzed in the oviducts of immature chicks during estrogen-promoted growth. Hybridization of oviduct total cellular RNA with viral-derived erb B oncogene probes demonstrated significant expression of c-erb B mRNA in oviduct cells of untreated chicks. Daily administration of estrogen (diethylstilbestrol) to chicks results in marked oviduct growth but did not appreciably affect expression levels of c-erb B messenger RNA in oviducts after 2, 4 or 6 days of treatment. Withdrawal of chicks from estrogen treatment resulted in termination of oviduct growth. However, c-erb B messenger RNAs were detectable in the nonproliferative tissue at 5 days after hormone withdrawal. Readministration of diethylstilbestrol, progesterone or diethylstilbestrol plus progesterone to hormone-withdrawn birds (secondary stimulation) also did not affect c-erb B messenger RNA levels in the oviduct. These results demonstrate significant expression of the cellular erb B (epidermal growth factor receptor) gene in the avian oviduct. However, EGF receptor messenger RNA synthesis is not modulated in the oviduct by steroid hormones.     

Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro

Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.     

In vitro canine oocyte nuclear maturation in homologous oviductal cell co-culture with hormone-supplemented media

The aim of the present research was to verify the influence of oviductal cell co-culture previously supplemented with steroids (estrogen, progesterone, or both) on IVM rates for oocytes from anestrous bitches that were cultured in vitro for 48, 72 and 96 h. Oocytes harvested from anestrous bitches were selected and allocated into four groups: Group 1 (co-culture in oviductal epithelial cells without hormonal supplementation-control); Group 2 (estrogen supplementation); Group 3 (progesterone supplementation); Group 4 (estrogen+progesterone supplementation). The oviductal epithelial cell culture was established 72 h prior to oocyte co-culture. After periods of 48, 72 and 96 h, the degree of oocyte nuclear maturation was assessed. Co-culture in oviductal epithelial cells with estrogen was not as beneficial for canine IVM as supplementation with progesterone and estrogen, or progesterone supplementation alone. Therefore, it was feasible to use co-culture with oviductal epithelial cells obtained from anestrous bitches for IVM (monolayer culture with oviduct cells previously supplemented with progesterone). Final stages of oocyte maturation were achieved at 72 and 96 h of culture; therefore, the duration of maturation for oocytes obtained from bitches in different stages of the estrous cycle should be taken into account.     

Sexual coloration, plasma concentrations of sex steroid hormones, and responses to courtship in the female keeled earless lizard (Holbrookia propinqua)

The reproductive condition, steroid hormone concentrations in the plasma, and behavior of bright and plain female Holbrookia propinqua were examined. Bright females performed significantly more aggressive rejection of courtship than plain females. Bright females were significantly more likely than plain females to have follicles larger than 5.0 mm or oviductal eggs; females with large follicles or oviductal eggs had significantly higher concentrations of progesterone and androgen than those with small follicles and lacking oviductal eggs. Plasma progesterone, androgen, and estradiol levels in the females studied behaviorally were significantly higher for the bright females than for the plain ones. Females undergoing rapid brightening were significantly more likely to be sexually receptive and copulate than were either plain or fully brightened females. The role of sex steroid hormones in coloration and behavior and the adaptive value of chromatic signalling by females are discussed.     

Estrogen and progesterone receptors in the oviduct during egg transport in cyclic and pregnant rats

We investigated the temporal relationships between ovum transport and changes in the concentration of nuclear steroid receptors in the oviduct of cyclic and pregnant rats. A lack of parallelism between estrogen and progesterone fluctuations in plasma and their respective nuclear receptor concentrations in the oviduct predominated during egg transport. In pregnant animals, oviductal egg transport took 24 h longer than in nonpregnant animals. In both conditions, transport was initiated while the action of estrogen and progesterone on the oviduct--measured as nuclear receptor accumulation--was decreasing. Three or four days later, depending on whether the animal was pregnant, the eggs entered the uterus shortly after an increase in the nuclear receptor accumulation of both hormones. Treatment with RU486, a progesterone receptor-blocking agent known to cause premature arrival of eggs in the uterus, advanced estrogen receptor accumulation in the oviduct of pregnant rats. These data suggest that the arrival of eggs in the uterus is timed by a transitory increase in nuclear estrogen receptor in the oviduct that does not necessarily reflect a similar change of circulating estradiol. Moreover, in pregnant rats, the onset of this estrogenic action is delayed by a progesterone receptor-mediated effect that hinders nuclear estrogen receptor accumulation.     

Magnal steroid hormone receptors during egg formation in the domestic hen

Levels of magnal estrogen and progesterone receptors during egg formation in the hen were determined. Hens were sacrificed at various times after ovulation and magnal receptor levels were determined by tritiated hormone binding assays. A coincident increase in nuclear estrogen receptor and decrease in cytosol estrogen receptor 2 to 4 h postoviposition was suggestive of in vivo receptor translocation. At 12 to 16 h postoviposition cytosol progesterone receptor increased 2-fold and subsequently declined during the time of preovulatory progesterone surge (8 h to 6 h prior to expected ovulation). These data suggest that changes in circulating levels of estrogen and progesterone, associated with ovulation, are coordinated with oviductal function. This is reflected by fluxes of their respective oviductal receptors.     

Co-culture of ovine eggs with oviductal cells and trophoblastic vesicles

Three experiments were conducted to determine whether coculture of early sheep eggs with oviductal cells would improve the ability of eggs to survive in culture. Eggs recovered from superovulated ewes were cultured in Ham's F-10 medium supplemented with 10% fetal calf serum (F10FCS) at 37.5 degrees C in 95% air:5% CO(2). In Experiment 1, eggs with one to eight cells were either transferred into recipient ewes immediately after collection or were cultured for 24 h in 5 ml Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS), 5 ml F10FCS on a confluent monolayer of oviductal cells; in 25 ml of fresh F10FCS; or in 25 ml of F10FCS removed cultures of oviductal cells, 25 mul of fresh F10FCS or 25 mul of F10FCS removed from cultures of oviductal cells. After 24 h, the cultured eggs were transferred to recipient ewes synchronous with donors and subsequently recovered at necropsy on Day 8 post estrus. Coculture of sheep eggs with oviductal cells improved (P < 0.05) the development of transferred eggs compared to culture in F10FCS alone. In Experiment 2, eggs recovered from superovulated ewes on Days 3 to 6 after estrus had undergone 1.8 cleavages by Day 3 and 4.1 cleavages by Day 6. In Experiment 3, single-cell eggs were cultured for 3 d in 5 ml F10FCS, cocultured with ovine trophoblastic vesicles or cultured on a confluent monolayer of oviductal cells. Coculture of eggs in F10FCS on a monolayer of oviductal cells supported in vitro egg cleavage to a greater degree than did F10FCS alone or F10FCS with trophoblastic vesicles (P < 0.05). In Experiment 4, single-cell eggs were cultured for 3 d then transferred to recipients. Eggs were cultured in 5 ml F10FCS on confluent monolayers of oviductal cells from luteal or estrous ewes or on cells that had been frozen after recovery from a culture of oviductal cells. After culture, the eggs were transferred to oviducts of recipients and recovered 3 d later at necropsy. Coculture of eggs for 72 h with oviductal cell monolayers did not increase the in vitro, or subsequent in vivo, cleavage rate regardless of the type of oviductal cells.     

Acceleration of oviductal transport of oocytes induced by estradiol in cycling rats is mediated by nongenomic stimulation of protein phosphorylation in the oviduct

In order to explore nongenomic actions of estradiol (E2) and progesterone (P4) in the oviduct, we determined the effect of E2 and P4 on oviductal protein phosphorylation. Rats on Day 1 of the cycle (C1) or pregnancy (P1) were treated with E2, P4, or E2 + P4, and 0.5 h or 2.5 h later their oviducts were incubated in medium with 32P-orthophosphate for 2 h. Oviducts were homogenized and proteins were separated by SDS-PAGE. Following autoradiography, protein bands were quantitated by densitometry. The phosphorylation of some proteins was increased by hormonal treatments, exhibiting steroid specificity and different individual time courses. Possible mediation of the E2 effect by mRNA synthesis or protein kinases A (PK-A) or C (PK-C) was then examined. Rats on C1 treated with E2 also received an intrabursal (i.b.) injection of alpha-amanitin (Am), or the PK inhibitors H-89 or GF 109203X, and 0.5 h later their oviducts were incubated as above plus the corresponding inhibitors in the medium. Increased incorporation of 32P into total oviductal protein induced by E2 was unchanged by Am, whereas it was completely suppressed by PK inhibitors. Local administration of H-89 was utilized to determine whether or not E2-induced egg transport acceleration requires protein phosphorylation. Rats on C1 or P1 were treated with E2 s.c. and H-89 i.b. The number and distribution of eggs in the genital tract assessed 24 h later showed that H-89 blocked the E2-induced oviductal egg loss in cyclic rats and had no effect in mated rats. It is concluded that E2 and P4 change the pattern of oviductal protein phosphorylation. Estradiol increases oviductal protein phosphorylation in cyclic rats due to a nongenomic action mediated by PK-A and PK-C. In the absence of mating, this action is essential for its oviductal transport accelerating effect. Mating changes the mechanism of action of E2 in the oviduct by waiving this nongenomic action as a requirement for E2-induced embryo transport acceleration.     

Oestrogen and progesterone concentrations in plasma and oviductal tissue of ewes exhibiting a natural or induced oestrus

Synchronisation of oestrus in Karagouniki ewes by administration of the standard dose of progesterone results in lower fertility than observed when these ewes ovulate naturally. This suggests that the optimum dose of progesterone may be breed dependent. The exogenous progesterone may perturb the concentrations of oestradiol-17beta and progesterone in blood plasma and the oviductal wall. This possibility was investigated using Karagouniki ewes allocated at random to three treatments (n=4 per treatment). Ewes were allowed to exhibit natural oestrus (N) or oestrus was synchronised by administration of 250 mg (LP) or 375 mg (HP) progesterone (subcutaneous implants) followed by PMSG at 8 mg/kg live weight i.m. 14 days later. Oestrus was observed using teaser rams. Blood samples were collected for plasma oestradiol-17beta and progesterone assay from the onset to the end of oestrus at 2 h intervals. The uterus of each ewe was recovered at the end of oestrus and samples of the oviductal wall were taken from both oviducts and prepared, separately, for progesterone and oestradiol-17beta assay. Statistical analysis was performed using univariate analysis of variance. Plasma oestradiol-17beta concentrations from the onset to the end of oestrus were highest for N ewes and lowest for HP ewes with the values for LP ewes occupying an intermediate position. The differences were significant (P<0.05) between HP and the other two treatments from 4 to 12 h after the onset of oestrus and then between all treatments until the end of oestrus. Plasma progesterone levels were similar and fairly constant from the onset to the end of oestrus for N and LP. The plasma progesterone levels for HP were significantly (P<0.05) higher than for the other two treatments throughout oestrus. In oviductal wall samples, the oestradiol-17beta concentration was significantly (P<0.05) higher for N ewes than for synchronised ewes and the levels were similar for LP and HP ewes. The concentration of oestradiol-17beta differed (P<0.05) between right and left oviducts for N ewes but not for ewes of either of the synchronised oestrus treatments. Progesterone concentrations in oviductal wall samples were highest (P<0.05) for HP ewes and the values for N and LP ewes were similar. The concentration of progesterone did not differ between right and left oviductal wall samples within treatments. It was concluded that the higher dose of exogenous progesterone perturbed the levels of oestradiol-17beta and progesterone in blood plasma and the oviductal wall, and this could explain the lower levels of fertility (relative to naturally occurring oestrus) observed when this protocol is used for Karagouniki ewes in practice.     

Effect of hormone treatments on chick oviduct β-N-acetylglucosaminidase isozymes and other acid hydrolases

The effects of several hormone treatments on chick oviduct acid hydrolases were studied; including the effect of those treatments on the isozymes of β-N-acetylglucosaminidase. Chicks were treated for 10 days with diethylstilbestrol after which they were treated with progesterone alone, diethylstilbestrol alone, progesterone and diethylstilbestrol, or withdrawn from all hormone treatment. Protease and acid phosphatase were increased four- to fivefold upon hormone withdrawal, but they were not increased by any of the other treatments. β-N-Acetylglucosaminidase, however, increased fourfold upon hormone withdrawal and progesterone alone or progesterone and diethylstilbestrol treatment. In addition to the increase in β-N-acetylglucosaminidase activity, isozyme II increased from 20 to 35% of the total activity upon withdrawal (but not the other treatments). Isozyme I is the only form of the enzyme found in egg white.     

Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells

The importance of glycoproteins located in the nuclear envelope in nuclear transport was tested by microinjection of karyophilic proteins into the cytoplasm of cultured human cells together with various lectins. Wheat germ agglutinin (WGA) blocked the nuclear transport of nucleoplasmin, a nuclear protein of Xenopus laevis oocytes, and of nonnuclear proteins conjugated with a synthetic peptide containing the nuclear localization signal sequence for simian virus 40 (SV40) large T antigen. Its inhibitory activity persisted for about 1 h after its injection into the cells and then gradually decreased. Export of at least some kinds of RNA from the nucleus seemed not to be affected by WGA even when import of the proteins into the nucleus was completely blocked (within 1 h after WGA injection). Moreover, WGA did not inhibit the passive diffusion of fluorescein isothiocyanate (FITC)-dextran (average Mr 17,900) into the nucleus. Wistaria floribunda agglutinin (WFA), concanavalin A (Con A), and lentil lectin did not block nuclear transport. These results indicate that WGA specifically blocks active protein import, but not passive diffusion of materials into the nucleus.     

Ciliary motility in bovine oviducts for sensing rapid non-genomic reactions upon exposure to progesterone.

Based on the expression of hormone receptors, oviductal cells receive a series of signals to control the conduit and transport of gametes. Most cells in the inner oviductal mucosa have motile cilia, and the mucociliary system of oviducts represents a prominent object of study for rapid feedback after application of steroid hormones. Using a high-speed reflectometry method, we investigated effects on the ciliary beat frequency (CBF) of bovine oviductal explants after progesterone treatment. To classify changes of CBF as either classic or non-classic reactions, we pretreated primary tissue cultures with mifepristone, an antagonist to the classic progesterone receptor in a second experimental series. In contrast to classical genomic reactions, non-classic or non-genomic reactions are characterized by fast effects, insensitive to the classic receptor antagonist. We observed inhibitory effects on the ciliary beat frequency as soon as 15 minutes after application of progesterone (20 microM), reaching a plateau of about 11 % after 90 minutes. Pretreatment with mifepristone (20 microM) for two hours did not induce significant differences in short-term reactions. However, the inhibitory influence of progesterone after 24 hours could be effectively prevented. Our data confirmed the short-term reaction of CBF as non-genomic or non-classic.     

Progesterone abbreviates the nuclear retention of estrogen receptor in the rat oviduct and counteracts estrogen action on egg transport

Progesterone has synergistic or antagonistic effects on several estrogenic actions. The effects of progesterone on estrogen-induced accelerated ovum transport and on the dynamics of estrogen receptors in the rat oviduct were examined. The involvement of the progesterone receptors in these phenomena was assessed. On Day 1 of pregnancy, rats were treated with estradiol, estradiol plus progesterone, or either one plus the progesterone receptor-blocking agent RU486. Control animals received the oil vehicle alone. The number of eggs remaining in the oviduct was assessed 24 h after treatment. Cytoplasmic and nuclear estrogen receptor levels in the oviduct, as well as plasma concentrations of estradiol and progesterone, were measured at various intervals--up to 11 h and 24 h after treatment, respectively. Accelerated oviductal egg transport induced by estrogen was blocked by the concomitant administration of progesterone. This effect of progesterone was not associated with changes in estrogen circulating levels and was preceded by a reduction in the total amount of estrogen receptors and by a shortened retention of estrogen receptors in the nucleus. The effects of progesterone on egg transport and on the levels of estrogen receptors were reversed by blocking the progesterone receptor with RU486, suggesting that both effects were receptor-mediated. These findings demonstrate that progesterone antagonizes the effect of estrogen on oviductal egg transport in the rat, and suggest that this antagonism is mediated by a reduction both in the amount of estrogen receptors and in their retention time in the nucleus.     

TGF-β_2在低氧条件下诱导骨髓基质干细胞向软骨细胞分化的研究

目的：探讨转化生长因子β2（TGF-β2）在低氧条件下诱导骨髓基质干细胞（BMSCs）向软骨细胞分化的作用。方法：无菌条件下分离Wistar大鼠股骨骨髓,采用全贴壁培养法纯化BMSCs。传6代后,将细胞随机分为3组,A组加入25 ng/mL TGF-β2在1%氧浓度条件下培养;B组加入25 ng/mL TGF-β2在21%氧浓度条件下培养;C组仅加入含10%胎牛血清的DMEM-α培养液在1%氧浓度条件下培养。3周后,通过甲苯胺蓝染色检测细胞糖胺多糖,聚合酶链反应检测Ⅱ型胶原和蛋白聚糖（Aggrecan）的表达水平。结果：骨髓细胞经换液后贴壁聚集生长,形态均一,连续传代后形态无明显改变。分组培养第1周,A、C组生长速度低于B组;第2周各组均出现不规则形态细胞,A、C组细胞形态小于B组;第3周各组均可见透明样基质,以A组最明显。3周后行甲苯胺蓝染色,A组细胞内外均可见丰富的蓝染颗粒,B、C组染色较A组略浅。A组Ⅱ型胶原的表达相对量（1.246±0.287）高于B组（0.973±0.365）、C组（0.802±0.196）,差异有统计学意义（P〈0.05）;B、C组比较无明显差异（P〉0.05）。A组Aggrecan的表达相对量（0.833±0.375）高于B组（0.724±0.173）、C组（0.602±0.091）,差异有统计学意义（P〈0.05）;B、C组比较无明显差异（P〉0.05）。结论：TGF-β2联合低氧环境可明显促进骨髓基质干细胞分化为软骨细胞。     

人颈椎间盘髓核细胞的体外培养和鉴定

目的：建立人颈椎间盘髓核细胞体外培养体系,并对其细胞表型进行鉴定。方法：采用酶消化法分离人颈椎间盘髓核细胞,进行单层培养,倒置相差显微镜观察细胞生长和形态,流式细胞仪测定细胞周期和凋亡率,并行甲苯胺蓝、Ⅱ型胶原及CK8免疫组化染色对其细胞表型进行鉴定。结果：原代髓核细胞凋亡率6.1±1.4%,S期细胞比例7.3±0.5%。贴壁后形态为多角形或短楔形,传代后生长加速。细胞呈甲苯胺蓝异染性;Ⅱ型胶原免疫组化染色阳性;只有少量椭圆形大细胞CK8免疫组化染色阳性。结论：成功建立人颈椎间盘髓核细胞体外培养模型,并证实成年后髓核内仍有少量细胞保持脊索细胞表型。