Do tensile and shear forces exerted on cells influence mechanotransduction through stored energy considerations?

All tissues in the body are subjected externally to gravity and internally by collagenfibril and cellular retractive forces that create stress and energy equilibrium required for homeostasis.Mechanotransduction involves mechanical work(force through a distance)and energy storage as kinetic and potential energy.This leads to changes in cell mitosis or apoptosis and the synthesis or loss of tissue components.It involves the application of energy directly to cells through integrin-mediated processes,cell-cell connections,stretching of the cell cytoplasm,and activation of the cell nucleus via yes-associated protein(YAP)and transcriptional coactivator with PDZ-motif(TAZ).These processes involve numerous complexes,intermediate molecules,and multiple pathways.Several pathways have been identified from research studies on vertebrate cell culture and from studies in invertebrates.These pathways involve mechanosensors and other molecules that activate the pathways.This review discusses the mitogen-activated protein kinase(MAPK)family,Hippo,Hedgehog,and Wingless-related integration site(WNT)/βcatenin signaling pathways.The mediators covered includeβcatenin,ion channels,growth factors,hormone receptors,members of the Ras superfamily,and components of the linker of nucleoskeleton and cytoskeleton(LINC)complex.However,the interrelationship among the different pathways remains to be clarified.Integrin-mediated mechanotransduction involves direct tensile loading and energy applied to the cell membrane via collagenfibril stretching.This energy is transferred between cells by stretching the cell-cell connections involving cadherins and the WNT/βcatenin pathway.These alterations induce changes in intracellular events in the cytoskeleton and nuclear skeleton caused by the release of YAP and TAZ.These coactivators then penetrate through the nuclear pores and influence nuclear cell function.Alteration in the balance of forces and energy applied to cells and tissues is hypothesized to shift the cell-extracellular matrix mechanical equilibrium by modifying mechanotransduction.The shift in equilibrium can lead to either tissue synthesis,genetic modifications,or promotefibrotic diseases,including epithelial cell-derived cancers,depending on the local metabolic conditions.

Associations of Gonadotropin-Releasing Hormone Receptor (GnRHR) and Neuropeptide Y(NPY) Genes’Polymorphisms with Egg-Laying Traits in Wenchang Chicken

Single nucleotide polymorphisms （SNP） of chicken gonadotropin-releasing hormone receptor （GnRHR） and neuropeptide Y （NPY） were selected to identify the genotypes of Wenchang （Chinese indigenous breed） chicken with restricton fragment length polymorphisms. The associations of the SNPs with the total egg production （NE）, average days of continual laying （ADCL）, and number of double-yolked eggs （DYE） traits were analyzed. The frequency of restriction enzyme A/a alleles in the population was for GnRHR 0.69 （Bpu1102 Ⅰ A） and 0.31 （Bpu1102 Ⅰ a） and for NPY 0.46 （Dra Ⅰ B） and 0.54 （Dra Ⅰ b）. Trait data from a total of 120 hens, which were purebred introduced from Hainan Province, China from one generation were recorded. Two significant effects of genes＇ marker were found： for GnRHR and number of eggs （dominant; t= 2.67, df= 116） and NPY and number of eggs （additive; t= 1.97, df= 116）. The current research supports the effects of GnRHR and NPY genes on egg-laying traits of chickens.

Thyroid hormones and thyroid hormone receptors: Effects of thyromimetics on reverse cholesterol transport

Reverse cholesterol transport (RCT) is a complex process which transfers cholesterol from peripheral cells to the liver for subsequent elimination from the body via feces. Thyroid hormones (THs) affect growth, develop- ment, and metabolism in almost all tissues. THs exert their actions by binding to thyroid hormone receptors (TRs). There are two major subtypes of TRs, TRα and TRβ, and several isoforms (e.g. TRα1, TRα2, TRβ1, and TRβ2). Activation of TRα1 affects heart rate, whereas activation of TRβ1 has positive effects on lipid and lipoprotein metabolism. Consequently, particular interest has been focused on the development of thyromimetic compounds targeting TRβ1, not only because of their ability to lower plasma cholesterol but also due their ability to stimulate RCT, at least in pre-clinical models. In this review we focus on THs, TRs, and on the effects of TRβ1-modulating thyromimetics on RCT in various animal models and in humans.

The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein （FSHR-57aa） as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a（＋）-FSHR-57aa plasmid was constructed and expressed in Escherichia coil strain BL21 StarTM （DE3） and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 （16 weeks） after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

Interactions Between Effects of Estrogen Receptor Gene Polymorphisms on BMD and Experiences of the First Spermorrhea in Chinese Han Boys

Objective To study the interaction between polymorphisms of estrogen receptor （ER） gene and puberty on bone mineral density （BMD）. Methods One hundred and forty-six boys aged 13-17 years were divided into two groups according to their first sperrnorrhea. DNA was analyzed for Xba I and Pvu 1/genotypes by PCR-RFLP. BMD of the total body, forearm and lumbar spine was measured by dual-energy X-ray absorptiometry （DXA）. The relationship between polymorphisms of ER gene and BMD in these two groups was analyzed. Results The BMD at all sites in the sperrnorrhea group was significantly higher than that in the un-sperrnorrhea group. The independent contribution of ER genotypes to BMD at two pubertal stages was analyzed after adjusting co-variables. In the un-sperrnorrhea group, the BMD at distal 1/10 and 1/3 forearm of those carrying pp genotype was significantly higher than that of the non-carries, whereas in the sperrnorrhea group BMD in those carrying the same genotype was significantly lower than that in the non-carriers. Similar results were obtained by haplotype analysis. Multiple stepwise regression analysis showed that body weight, age and the first sperrnorrehea were the dominant determinants for BMD. BMD at forearm might be influenced by interaction between ER genotype and the first spermorrehea. Conclusion The polymorphisms of ER gene play a different role in BMD influenced by the first spermorrhea. Chinese boys carrying p or x allele should pay more attention to their bone mass.

Fluoride Exposure,Follicle Stimulating Hormone Receptor Gene Polymorphism and Hypothalamus-pituitary-ovarian Axis Hormones in Chinese Women

The effects of fluoride exposure on thefunctions of reproductive and endocrine systemshave attracted widespread attention in academiccircle nowadays. However, it is unclear whether thegene-environment interaction may modify thesecretion and activity of hypothalamus-pituitary-ovarian （HPO） axis hormones. Thus, the aim of thisstudy was to explore the influence of fluorideexposure and follicle stimulating hormone receptor（FSHR） gene polymorphism on reproductivehormones in Chinese women. A cross sectionalstudy was conducted in seven villages of HenanProvince, China during 2010-2011. A total of 679women aged 18-48 years were recruited throughcluster sampling and divided into three groups, i.e.endemic fluorosis group （EFG）, defluoridationproject group （DFPG）, and control group （CG） basedon the local fluoride concentration in drinkingwater. The serum levels of gonadotropin releasinghormone （GnRH）, follicle stimulating hormone（FSH）, luteinizing hormone （LH）, and estradiol （E2）were determined respectively and the FSHRpolymorphism was detected by real time PCR assay.The results provided the preliminary evidenceindicating the gene-environment interaction onHPO axishormones in women.

Effect of neoadjuvant chemotherapy on the expression of hormone receptors and Ki67 in Chinese breast cancer patients:A retrospective study of 525 patients

This study was designed to investigate the effect of neoadjuvant chemotherapy on the expression of hormone receptors and Ki67 in Chinese female breast cancer patients. The expression of estrogen receptor（ER）, progesterone receptor（PR） and Ki67 among 525 neoadjuvant chemotherapy cases was studied by immunohistochemistry.Differences between specimens made through preoperative core needle biopsy and excised tissue biopsy were observed. The positive rates of ER, PR and Ki67 in core needle biopsy and excised tissue biopsy were 65.3% and 63.2%, 51.0% and 42.6%, 65.6% and 43.4%, respectively. The expression of ER, PR and Ki67 in core needle biopsy and excised tissue biopsy had no statistically significant difference. However, after neoadjuvant chemotherapy, the discordance rates of ER, PR and Ki67 were 15.2%（79/521）, 26.9%（140/520） and 44.8%（225/502）, respectively. The ER, PR and Ki67 status changed from positive to negative in 7.5%（39/521）, 13.3%（69/520） and 21.1%（106/502） of the patients, whereas ER, PR and Ki67 status changed from negative to positive in 7.7%（40/521）, 13.6%（71/520）and 23.7%（119/502） of the patients, respectively. These results showed that the status of some biomarkers changes after neoadjuvant chemotherapy and biomarker status needs to be reexamined to optimize adjuvant systemic therapy and better prognosis assessment.

Predictors for partial suppression of spermatogenesis of hormonal male contraception

Aim： To analyze factors influencing the efficacy of hormonal suppression of spermatogenesis for male contraception. Methods： A nested case-control study was conducted, involving 43 subjects, who did not achieve azoospermia or severe oligozoospermia when given monthly injections of 500 mg testosterone undecanoate （TU）, defined as partial suppressors compared with 855 subjects who had suppressed spermatogenesis （complete suppressors）. Sperm density, serum testosterone, luteinizing hormone （LH） and follicle stimulating hormone （FSH） concentrations at the baseline and the suppression phase were compared between partial and complete suppressors. Polymorphisms of androgen receptor （AR） and three single nucleotide variants and their haplotypes of FSH receptor （FSHR） genes determined by polymerase chain reaction （PCR） and DNA sequencing technique were compared between 29 partial and 34 complete suppressors. Results： Baseline serum LH level was higher and serum LH as well as FSH level during the suppression phase was less suppressed in partial suppressors. Additionally, in a logistic regression analysis larger testis volume, higher serum FSH concentrations alone, or interaction of serum LH, FSH, testosterone and sperm concentrations were associated with degree of suppression. The distribution of polymorphisms of AR or FSH receptor genes did not differ between partial and complete suppressors. In cases with incomplete FSH suppression （FSH 〉 0.2 IU/L）, the chances of reaching azoospermia were 1.5 times higher in the subjects with more than 22 CAG triplet repeats. Conclusion： Partial suppression of spermatogenesis induced by 500 mg TU monthly injections is weakly influenced by hormonal and clinical features but not polymorphism in AR and FSHR genes.

Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of humanFSH receptor promoter

<abstract>Aim: To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function. Methods: We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5' flanking region fragment of its promoter. Results: Mice were phenotypically normal and fertile. In males, mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic litter mates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells, spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5 '-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

Cloning and Transcriptional Activity of Follicle-Stimulating Hormone Receptor Promoter in the Jintang Black Goat

[ Objective] To clone follicle-stimulating hormone receptor （FSHR） promoter in the Jintang black goat, study its transcriptional activity, and provide a basis for alternative splicing of FSHR gene. [Method] The total DNA were extracted from the womb of Jintang black goat, and one pair of primers were designed for amplification of FSHR promoter fragments, then the sequences and homology were analyzed. The FSHR promoter fragment was inserted into the pcFSHRB1 expression vector to substitute the CMV promoter and construct the pcFSHRB2 expression vector. The pcFSHRB1 and pcFSHRB2 expression vectors were transformed into HEK293 cells, respectively. Then these cells were collected after 24 and 48 h treatment with 2 mlU/ml follicle-stimulating hormone （FSH）, and the cAMP levels were detected. [Result] The FSHR promoter sequence of Jin- tang black goat had 34.2% homology to that of chicken and 41.6% to that of rat, respectively. The transcription initial site of FSHR was at -576 bp and its upstream sequences contained two TATA-boxes, four CAAT-boxes, one E-box and one Wl-box. After treating for 24 and 48 h, the cAMP levels of pcFSHRB2 were respectively 299.581 3 and 125.528 1 pmol/L; and that of pcFSHRB1 were respectively 120.057 1 and 109.940 7 pmoVL. [Conclusion] The FSHR promoter of Jintang black goat is a typical type 2 eukaryotic promoter, and it is also a strong promoter.

Expression of anti-Mullerian hormone receptor on the appendix testis in connection with urological disorders

The female internal sex organs develop from the paramesonephric （Mullerian） duct. In male embryos, the regression of the Mullerian duct is caused by the anti-Mullerian hormone （AMH）, which plays an important role in the process of testicular descent. The physiological remnant of the Mullerian duct in males is the appendix testis （AT）. In our previous study, we presented evidence for the decreased incidence of AT in cryptorchidism with intraoperative surgery. In this report, the expression of the anti-Mullerian hormone receptor type 2 （AMHR2）, the specific receptor of AMH, on the AT was investigated in connection with different urological disorders, such as hernia inguinalis, torsion of AT, cysta epididymis, varicocele, hydrocele testis and various forms of undescended testis. The correlation between the age of the patients and the expression of the AMHR2 was also examined. Reverse transcriptase-polymerase chain reaction （RT-PCR） and immunohistochemistry were used to detect the receptor＇s mRNA and protein levels, respectively. We demonstrate that AMHR2 is expressed in the ATs. Additionally, the presence of this receptor was proven at the mRNA and protein levels. The expression pattern of the receptor correlated with neither the examined urological disorders nor the age of the patients; therefore, the function of the AT remains obscure.

Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide

BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR type 1(PTHR1)activates events associated with an aggressive phenotype.In HCT116 cell xenografts,PTHrP modulates the expression of molecular markers linked to tumor progression.Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC.Based on these data,we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.AIM To elucidate the relationship among PTHR1,PTHrP,and Met in CRC models.METHODS For in vitro assays,HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP(1-34)(10-8 M).Where indicated,cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide,the vehicle of the inhibitors.The protein levels were evaluated by Western blot technique.Real-time polymerase chain reaction(RT-qPCR)was carried out to determine the changes in gene expression.Wound healing assay and morpho logical monitoring were performed to evaluate cell migration and changes related to the epithelialmesenchymal transition(EMT),respectively.The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan(CPT-11),oxaliplatin(OXA),or doxorubicin(DOXO)with or without PTHrP.For in vivo tests,HCT116 cell xenografts on 6-wk-old male N:NIH(S)_nu mice received daily intratumoral injections of PTHrP(40μg/kg)in 100μL phosphate-buffered saline(PBS)or the vehicle(PBS)as a control during 20 d.Humanitarian slaughter was carried out and the tumors were removed,weighed,and fixed in a 4%formaldehyde solution for subsequent treatment by immunoassays.To evaluate the expression of molecular markers in human tumor samples,we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr.JoséPenna(Bahía Blanca,Buenos Aires,Argentina)and the Hospital Provincial de Neuquén(Neuquén,Neuquén,Argentina)from January 1990 to December 2007.Seven cases with normal colorectal tissues were assigned to the control group.Tumor tissue samples and clinical histories of patients were analyzed.Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique;subsequently,representative histological samples were selected from each patient.From each paraffin block,tumor sections were stained for immunohistochemical detection.The statistical significance of differences was analyzed using proper statistical analysis.The results were considered statistically significant at P<0.05.RESULTS By Western blot analysis and using total Met antibody,we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells.In HCT116 cells,Met protein levels increased at 30 min(P<0.01)and at 20 h(P<0.01)whereas the levels diminished at 3 min(P<0.05),10 min(P<0.01),and 1 h to 5 h(P<0.01)of PTHrP treatment.Using an active Met antibody,we found that where the protein levels of total Met decreased(3 min,10 min,and 60 min of PTHrP exposure),the status of phosphorylated/activated Met increased(P<0.01)at the same time,suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP.The increment of its protein level after these decreases(at 30 min and 20 h)suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis(P<0.05).We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/activation of Met induced by PTHrP in HCT116 cells.By Western blot technique,we observed that PP1,a specific inhibitor of the activation of the protooncogene protein tyrosine kinase Src,blocked the effect of PTHrP on Met phosphorylation(P<0.05).Furthermore,the selective inhibition of the ERK 1/2 mitogen-activated protein kinase(ERK 1/2 MAPK)using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation(P<0.05).Using SU11274,the specific inhibitor of Met activation,and trypan blue dye exclusion test,Western blot,wound healing assay,and morphological analysis with a microscope,we observed the reversal of cell events induced by PTHrP such as cell proliferation(P<0.05),migration(P<0.05),and the EMT program(P<0.01)in HCT116 cells.Also,PTHrP favored the chemoresistance to CPT-11(P<0.001),OXA(P<0.01),and DOXO(P<0.01)through the Met pathway.Taken together,these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells.By immunohistochemical analysis,we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met(0.190±0.014)compared to tumors from control mice(0.110±0.012;P<0.05)and of its own receptor(2.27±0.20)compared to tumors from control mice(1.98±0.14;P<0.01).Finally,assuming that the changes in the expression of PTHrP and its receptor are directly correlated,we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis.Comparing histologically differentiated tumors with respect to those less differentiated,we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner,respectively(P<0.05).CONCLUSION PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model.More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.

Molecular Characterization of Thyroid Hormone Receptors (TRs) and their Responsiveness to T3 in Microhylafissipes

To explore and enrich the molecular mechanisms of thyroid hormone receptors （TRs） in the metamorphosis of amphibians, the cDNA sequences of TRa and TRβ in Microhyla fissipes were cloned and characterized. TRa was 1 706 bp in length with an open reading frame （ORF） of 1 257 bp encoding a predicted protein of 418 amino acids and TRβ was 1 422 bp with an ORF of 1 122 bp encoding a predicted protein of 373 amino acids. Their protein sequences contained 4 conserved domains of the nuclear receptor superfamily with two highly conserved cysteine-rich zinc fingers in the DNA-binding domain, whereas TRβ was 42 amino acids shorter in its A/B domain than TRot. Highly-conserved sequences and structures indicated their conserved functions during metamorphosis. TRa expression reached peak at 12 h and then decreased from 12 h to 48 h. While dramatically up-regulated TRβ was observed after exposure of T3 within 24 h, and it was down-regulated from 24 h to 48 h. The expression pattern of TRβ is similar to that in the natural metamorphosis. Furthermore, tadpoles treated 24 h also resembled the climax of metamorphosis tadpoles and TRβ expression had higher responsiveness than TRa to T3 in M. fissipes. These results suggest M. fissipes may serve as the model to assay environmental compounds on TH signaling disruption.

Growth hormone receptor expression in human primary gastric adenocarcinoma

The aim of this study was to determine the expression of growth hormone receptor（GHR） in patients with pri-mary gastric adenocarcinoma.We investigated 48 specimens of primary gastric adenocarcinoma and their cor-responding normal gastric mucosa.Immunohistochemistry and reverse transcription-polymerase chain reaction（RT-PCR） were used to detect the expression of GHR.Immunohistochemical analyses revealed that GHR was expressed in human primary gastric adenocarcinoma（36/48,75.0%） and appeared to be upregulated,compared to the normal mucosa（28/48,58.3%,P 〈 0.001）.A significant correlation was found between GHR expression and tumor stage（P 〈 0.001） and tumor differentiation（P 〈 0.001）.The average positive rate of ki-67 in GHR-positive tumors was 16.06%,while the positive rate in GHR-negative tumors was 6.17%（P 〈 0.01）.The average apoptosis index（AI） of GHR-positive tumors was 3.36%,which was significantly lower than that（7.33%） of GHR-negative tumors.In addition,27 of 48 cases of tumors had GHR mRNA expression,while only 17 of all 48 cases of normal mucosa did so.Our results indicate that the frequency of GHR was significantly higher in primary gastric adeno-carcinoma than that in normal gastric mucosa.GHR expression was significantly correlated with tumor differen-tiation and tumor grade.This finding supported a possible role of growth hormone in primary gastric adenocarci-noma pathophysiology.

Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Cell Proliferation and Apoptosis in Cells Derived from Human Ovarian Mucinous Cystadenocarcinoma in Vitro

The human ovarian mucinous cystadenocarcinoma （hOMC） cells were co-cultured with antisense oligodeoxynucleotide （antisense ODN）, nonsense ODN, and follicle-stimulating hormone （FSH） at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor （FSHR） on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups （P〈0.05 or P〈0.01）, decreased distinctly in antisense ODN groups （P〈0.05 or P〈0.01）, and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity （P〈0.01）. Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid- and high-dose antisense ODN groups （P〈0.05 or P〈0.01）, while the number of cells in G1/G0 phase was significantly decreased and that in S phase distinctly increased （P〈0.01）, There was no change in nonsense ODN groups （P〉0.05）, It was suggested that FSH may improve the development of hOMC cells, However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis.

CORRELATION OF STEROID HORMONE RECEPTORS AND CLINICAL PATHOLOGICAL FEATURES WITH PROGNOSIS OF HUMAN BREAST CANCER

Clinical, pathological features and steroid hormone receptors (SR) including receptors of estrogen (ER), progesterone (PR) and androgen (AR) were observed in 58 cases of breast carcinoma, and related to patient 5- year survival rate through stratification and multivariatc analysis. The results showed that histologic tumor type and grading, lymphnode status, ER value and patient age took more important role in patient survival, and SR, especially, conferred survival advantage in advanced cases with tumor size larger than 2 cm, node involved, or TNM Stage Ⅱ-Ⅲ.

Immunohistochemical Localization and Receptor Expression of Gonadotropin-Releasing Hormone in Pituitary of Guangxi Swamp Buffaloes

[ Objective] To locate gonadotropin-releasing hormone （GnRH） in pituitary of Guangxi swamp buffaloes and to provide a theoretical ba- sis for cloning and sequence analysis of GnRH receptor gene. [ Method] GnRH in pituitary were immunohistochemically stained by avidin biotin complex method. The GnRH expression was analyzed with image system. The GnRH receptor gene was amplified by real-time PCR. [ Result] Many GnRH positive cells were detected in pars distalis of adenohypophysis. GnRH were distributed in cytoplasm but not in nuclei. No positive sig- nal was observed in neurohypophysis. In addition, the GnRH receptor gene, 920 bp in size, was amplified. [ Conclusion] A large number of GnRH and GnRH receptor were found in pars distalis of adenohypophysis, which indicates that anterior pituitary is an important tissue for functions of hypo- thalamus-pituitary-gonadal axis and other endocrine axes.

STUDY OF ESTROGEN RECEPTOR AND PROGESTERONE RECEPTOR IN SQUAMOUS CELL CARCINOMA TISSUE OF ORAL MUCOSA

By means of the enzyme linked affinity histothemical method, 45 cases of squamous cell carcinoma(SCC) of the oral mucosa and 15 cases of the approximately normal oral mucosal tissue were detected for estrogen receptor(ER) and progesterone receptor(PR). The results indicated that In the SCC tissue of the oral mucosa there were 5 cases of ER+ and PR- and 32 cases of both ER+ and PR+. Thirty-seven cases, the summation of the above two items, were considered as receptor(+),therefore the rate of the receptor(+) being 82.22%. ER+ and PR+ were cd related to the sex and age of patients, the neck lymph nodes' metastasizing or not and affected parts of the tumor, while they were related to the differentiation degree of the tumor. The rate of receptor(+) decreased with the decrease of the differentiation degree of the tumor. By X2 test a remarkable difference between grade Ⅰand grade Ⅲ of SCC of the oral mucosa was shown. It is suggested that SCC of the oral mucosa may probobly be hormone dependent tumor. The authors consider that the SCC detection for ER and PR could not only be one of the indices of biologic characteristics for that tumor but also as bases of anti-hormone treatment.

EXPRESSIONS OF ESTROGEN OCCUPIED RECEPTOR（EoR） AND PROGESTERONEOCCUPIED RECEPTOR（PoR） AND C－erbB－2ONCOPROTEININ HUMANNASOPHAR

It is first reported here that estrogen occupied receptor(EoR)and progesterone occupied receptor (RoR)expressed in cancerous tissues (59.57% and 82.98% respectively)and morphologically normal epithlium(50 77.78% and 70-88.89%respectively) in nasoplharyngeal carcinomas(NPCs)with insignificant difference(P>0.05).Positive rates of EoR and PoR increased greatly in clinical stage Ⅲ and Ⅳ, compared with in Ⅱ(P<0.05), and exhibited insignificant difference between female cases and male ones(P>0.05).Positive rate of C-erbB-2 was 19.15% in cancerous cells, and 9.68% in stage Ⅲand 66.67% in Ⅳin NPCs(P<0.05).Significant difference of C-erbB-2expression was observed between bilateral cervical lymph node metastasis(BCLM)and unilateral ones(P<0.05)but not for EoR or PoR(P>0.05)These findings suggest that EoR or PoR may be correlated with aggravation but not genesis and node metastasis in NPCs and that C-erbB-2may be correlated with aggravation and promotion of formation of node metastasis in NPCs.