【作者】 白艳艳；

【导师】 张源潮； Michael Underill；

【作者基本信息】 山东大学 ， 内科学， 2013， 博士

【摘要】 研究背景系统性红斑狼疮(systemic lupus erythematosus,SLE)是以多种自身抗体及多样性的多个系统和器官的炎症性损害为特征的系统性自身免疫病。虽然确切的病因和发病机制尚不清楚,但多种遗传和环境因素及免疫学异常可能参与其中。既往研究发现SLE患者存在T细胞和自然杀伤(natural killer, NK)细胞的异常活化及NK细胞数量减少和杀伤活性降低。并且SLE患者中CD3+CD56+natural killer T (NKT)细胞也存在数量减少现象并与SLE患者中高水平IgG及IgG anti-dsDNA抗体相关。因此可推测淋巴细胞亚群失衡与SLE发病相关。杀伤细胞免疫球蛋白样受体(Killer cell immunoglobulin-like receptors, KIR)为含有I型跨膜糖蛋白的免疫球蛋白超家族(immunoglobulin superfamily, IgSF)进化而来,表达于自然杀伤细胞及部分T细胞表面。KIR基因具有多态性及多基因性的特点。近期研究显示NK-和T-细胞KIR的表达模式为KIR基因位点甲基化状态所调控。已发现14个KIR基因和2个假基因。KIR分子的胞外段有两个(2D)或三个(3D)免疫球蛋白样结构域,而胞内段分为长(L)和短(S)两种类型。抑制性KIRs的长胞内段含有免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif, ITIM),而活化性KIRs则为相对较短的胞内段,由含有adaptor DAP12的酪氨酸激活基序构成。KIRs的配体为人类白细胞抗原(human leukocyte antigen, HLA)I类分子,并且,不同的KIR和HLA组合,可以发挥不同的功能,抑制或活化NK细胞的杀伤活性或炎性细胞因子的分泌。基因学研究显示KIR和很多疾病状态存在广泛的相关性,包括感染性疾病如自身免疫缺陷病毒(HIV)和丙肝病毒(HCV)引起的疾病,自身免疫及炎症性异常如硬皮病、银屑病关节炎,癌症如宫颈癌,还有先兆子痫。一般来说,低抑制高活化的基因型模式有益于清除病毒性感染,但该群体患自身免疫性疾病的风险性却是增加的。而抑制性KIR显著的基因型似乎是先兆子痫的高风险因素之一。Pellet等发现在无KIR2DS2的情况下KIR2DS1基因频率在SLE患者中高于健康对照组。并且发现发生过血管性事件的患者较未发生的患者存在显著性增高的KIR2DS2及KIR2DL2基因频率。我们前期试验也发现中国SLE患者中KIR2DL2和KIR2DS1基因型频率增加且含有两个以上活化性KIR的基因型相对于健康对照组来说更常见于患者组。狼疮患者的T细胞存在去甲基化状态,使得活化性和抑制性KIRs在SLE患者中T细胞表达并具功能。因用于做流式检测的KIR2DL3/L2/S2和KIR2DL1/S1的抗体在未进行基因型检测的基础上无法区分活化性和抑制性KIRs,因此关于该领域的研究目前尚无明确结论。研究目的探明杀伤细胞免疫球蛋白样受体在系统性红斑狼疮患者NK(CD3-CD56+)细胞,natural killer T (NKT)(CD3+CD56+)细胞及T (CD3+CD56-)细胞上的表达模式特征。分析SLE患者CD158a/h, CD158b/i/j在NK-、NKT-、T-细胞上的表达与SLEDAI评分及各种免疫性特征之间是否存在相关性。并探明KIR2DL1, KIR2DS1及KIR2DL2在SLE患者NK-、NKT-、T-细胞中的转录情况。从而综合分析KIR在SLE患者淋巴细胞表达模式与发病机制的相关性。研究方法1.筛选SLE患者和健康对照SLE患者和健康对照均分为2DS1+2DL1+组(SLE患者组：n=16；健康对照组：n=12)和2DS2-2DL2+2DL3+组(SLE患者组：n=12；健康对照组：n=8)。SLE患者的免疫学指标数据由医院临床实验室提供。血清IgG> IgA> IgM、C3及C4水平通过Scatter Turbidimeter检测；IIFT法检测ANA,血清ds-DNA、 ss-DNA、AnuA、AHA、rRNP和SM通过ELISA法检测。样本各KIR基因的存在与否通过PCR-SSP方法测得。2.流式细胞术分析分离PBMC应用EPICS XL-4进行流式分析。所用主要单克隆抗体如下：(i)anti-human CD3(PE),(ii) anti-human CD56(Cy5),(iii) FITC-CD158a/h (EB6)(KIR2DL1/KIR2DS1),(iv) FITC-CD158b/i/j (GL183)(KIR2DL2/KIR2DL3/KIR2DS2)及各特异性抗体的同型对照.3.应用Pearson correlation coefficient (r2)分析CD158a/h, CD158b/i/j在SLE患者NK-、NKT-、T-细胞上的表达与SLEDAI评分及各种免疫性特征之间的相关性。two-tailed p-value≤0.05被认为具有统计学意义。4.应用CD56multi-sort试剂盒和CD3免疫磁珠分选SLE患者组及健康对照组PBMCs中的NK-、NKT-和T细胞。应用ABI PRISM7500检测系统通过RT-PCI技术以18S基因为对照分析KIR2DL1、KIR2DS1、KIR2DL2在NK-、NKT-和T细胞中的转录水平。实验结果1.SLE患者NK细胞在PBMC中的比率显著低于健康对照组(p<0.001)。同时,相对于健康对照组SLE患者的NKT细胞在外周血单个核细胞中的比率亦降低(p<0.001)。但是T细胞比率在两组中无显著差异。2. CD158a/h在SLE患者NK细胞的表达相对于健康对照组显著增加(p<0.001)。而相对于健康对照组CD158b/i/j在SLE患者NK细胞的表达显著降低(p<0.001)。并且SLE患者NK细胞CD158b/i/jMFI显著增高(p=0.036)。3.SLE患者NKT细胞CD158a/h表达显著高于健康对照组(p<0.001)。而CD158b/i/j在该类型细胞中的表达则是SLE患者组显著低于健康对照组(p=0.002)。4.SLE患者T细胞CD158a/h表达显著增加(p=0.026)。5.SLE患者NK细胞CD158a/h的表达与血清anti-dsDNA水平成正相关(R=0.742,p=0.001)。6.SLE患者CD158bl/b2/j+NKT细胞PBMCs中的比率与血清IgG水平成负相关(R=-0.591,p=0.043)7.相对于健康对照组SLE患者NK细胞KIR2DS1转录显著增加(p=0.025)而KIR2DL1转录显著减少(p=0.049)。8.相对于健康对照组KIR2DS1的转录在SLE患者NKT细胞中显著上调(p=0.041)。结论1.我们的研究结果证实SLE患者NK细胞活化性KIR表达增加而抑制性KIR表达减少。KIR2DS1的表达增加可能会降低SLE患者NK细胞对于靶细胞(例如自身反应性T细胞)的杀伤效应,从而导致自身抗体(例如Anti-dsDNA抗体)的生成。所以KIR在SLE患者NK细胞上的异常表达模式可能会影响此类型细胞的分化和功能活性并可能因此参与SLE的发病。2. KIRs在NKT细胞的异常表达可能具有功能并参与SLE的发病,并且可能至少部分是通过影响血清IgG水平实现的。3.狼疮患者T细胞上表达的活化性和抑制性KIRs是功能性的,而过表达的活性性KIRs可能参与了SLE患者自身反应性T细胞的活化。4. CD158a/h+NK-、NKT-和T细胞比率在SLE患者组是增加的,而它们的MFI水平却无显著性差异。因此KIR2DS1mRNA的增加可能是2DS1+淋巴细胞普遍扩增所致,而不是由少量很高表达2DS1的NK或NKT细胞亚群所致。研究背景：反应性关节炎(reactive arthritis, ReA)是一种由细菌性炎性肠病或泌尿生殖系统感染引起的无菌性关节炎。虽然ReA的发病机制尚未明确,但遗传因素似乎在其中具有重要作用。不同的杀伤细胞免疫球蛋白样受体(KIRs)和它们相对应的特异性组织相容性白细胞抗原-C (HLA-C)配体基因型组合在感染性疾病及自身免疫性疾病的易感性和耐受性方面的相关性研究被广泛探讨,但迄今为止,尚无关于ReA的相关研究。方法：本次实验纳入138例ReA患者(女性65例,男性73例)；年龄18至69岁(平均37岁)和种族、年龄和性别相匹配的151例随机筛选的健康对照。样本均采用序列特异性聚合酶链式反应方法对KIR基因和HLA-C等位基因的基因型进行检测。结果：ReA患者中抑制性的KIR2DL2和KIR2DL5基因频率显著低于对照组(分别为P=0.005和P=0.033)。含有多于7个抑制性KIR的基因型不易发生ReA (P=0.016)。并且,我们发现活化性的KIR2DS1单独或和HLA-C1C1组合(即表示其同源抑制性基因KIR2DL1相对应的配体缺失)的基因型为ReA的易感因素(分别为P=0.039和P=0.011)。而KIR2DL2和它的配体HLA-C1同时存在却对个体起到保护作用,使其不易于患ReA(P=0.039).结论本次研究提示高水平的活化性和低水平的抑制性KIR信号可能会影响NK细胞和T细胞的功能,而这种固有和适应性免疫反应的失衡导致个体易于被致病原所激发,导致局部的或系统性的细胞因子生成过多从而引起ReA的发病。研究背景：肿瘤超甲基化基因1(Hypermethylated in cancer1, HIC1),是定位于17p13.3的转录阻遏物,存在于一个CpG岛中,在很多种癌症中呈高甲基化状态,与细胞增生、肿瘤生长和血管生成相关。最近,Underhill课题组构建出一种新的条件性HIC1基因敲除小鼠模型。对这种鼠的初步分析显示这种基因的缺陷导致多种骨骼畸形,包括骨的形态学异常,颅缝早闭及骨盐密度减低。我们发现这种条件性HIC1基因敲除小鼠胚胎唾液腺发育异常,所以本次实验拟探讨其发生机制以更好的了解HIC1的作用机制。并且这项研究给了我们一个应用此新的动物模型研究唾液腺异常性疾病的发病及治疗的机会。方法：1.取不同胎龄的小鼠胚胎,分离SMG并采集图像,同时检测其基因型。2.提取模型小鼠胚胎SMG RNA进行quantitative real-time PCR,检测BTBD7、 FGF10、MMP2、MMP9、Fnl (fibronectin)、Inhibin beta A (Activin beta A)、 Inhibin beta B (Activin beta A)的转录水平。3.模型小鼠SMG器官培养和Whole-mount X-gal染色。分离E13.5胎鼠SMGs滤膜上培养过夜后进行X-gal染色及图像采集。4.免疫组化和免疫荧光。模型小鼠胚胎组织甲醛固定脱水,石蜡包埋切片(厚度7mm),进行H&E及特异性抗体的免疫组化和免疫荧光染色。所用抗体包括：anti-Fnl (fibronectin、anti-colkligenl、anti-Ki67、anti-AQP5。二抗与488Alexa-fluor或594Alexa-fluor联合。结果：1.HIC1-/-胎鼠SMG由分化不全的较少分支的上皮组织和紧密排列分布的间充质构成。2.HIC1-/-胎鼠SMG AQP5表达减少。3.HIC1表达分布于SMG间充质,上皮组织无分布。4.HIC1-/-胎鼠SMG间充质细胞增生增加。5.HIC1-/-胎鼠SMG发育晚期阶段Fibronectin表达减少。6.HIC1-/-胎鼠SMG发育晚期阶段Collagen I表达减少。7.HIC1-/-胎鼠SMG E15.5胎龄时FGF-10转录减少。8.HIC1-/-胎鼠SMG E14.5胎龄时MMP2和MMP9转录增加。9. Activin beta A和Activin beta B的转录在HIC1-/-胎鼠SMGE14.5时增加。结论1.HIC1在SMG形态发育中具有重要作用,缺失该基因时SMG发育迟滞,分支状态和体积均异常。2.HIC1在SMG功能发育分化中具有重要作用,缺失该基因时作为分泌功能标志物的AQP5表达减少。3.HIC1定位于SMG间充质,其可能通过影响间充质细胞的增生间接影响了SMG功能发育分化。4.HIC1可能通过减少Fibronectin的表达影响SMG发育后期间叶结构的形成。5. Collagen I、TGF10、MMP2和MMP9可能也部分参与了HIC1信号系统对胎鼠SMG发育的调节。更多还原

【Abstract】 BackgroundSystemic lupus erythematosus (SLE) is a systemic auto-immune disorder,which is characterized by the production of different auto-antibodies and diverse clinical inflammation in multiple organ systems. Although the precise etiology and pathogenesis of SLE still remains unclear, various genetic and environmental factors along with multiple immunological abnormalities may be involved. Previous studies suggested the aberrant activation of T-cells and natural killer (NK) cellsith fewer NK cells and decreased cytotoxicity of NK cells in SLE patients. Furthermore, a lower frequency of CD3+CD56+natural killer T (NKT) cells was found to be associated with higher levels of plasma IgG and IgG anti-dsDNA antibodies in SLE patients. Hence, an imbalance of lymphocyte subsets has been implicated in the pathogenesis of SLE.Killer cell immunoglobulin-like receptors (KIR), which evolved from the Ig superfamily (IgSF) consisting of type I transmembrane glycoproteins, are expressed on NK cells and subsets of T-cells. The KIR genes are polymorphic, whereas the KIR gene complex is polygenic. Recent studies showed that the expression patterns of KIR on NK-and T-cells were regulated by the methylation of KIR gene loci. Fourteen KIR genes and two pseudogenes have been described. The extracellular domains of KIR molecules contain either two (2D) or three (3D) Ig-like domains, while the cytoplasmic tails are either long (L) or short (S). The inhibitory KIRs possess a long cytoplasmic tail, which contains immunoreceptor tyrosine-based inhibitory motif (ITIM), while the stimulatory KIRs possess a relatively shorter cytoplasmic tail, which comprises of tyrosine-based activation motifs containing adaptor DAP12. Human leukocyte antigen (HLA) class I molecules serve as ligands for the KIRs; besides, with different KIR and HLA, it either stimulates or inhibits the killing and secretion of inflammatory cytokines of NK cells.Genetic studies indicated the existence of an association between KIR and infectious diseases such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), autoimmune and inflammatory disorders such as scleroderma and psoriatic arthritis, cancer such as cervical cancer, and pre-eclampsia. Generally, the genotypes with lower inhibition and higher activation patterns appear to be beneficial in viral infections, but they constitute a risk for susceptibility to auto-immunity. However, the genotypes in which inhibitory KIR predominates seems to be at an increased risk of pre-eclampsia.According to Pellet et al., gene frequency of KIR2DS1in the absence of KIR2DS2increases in SLE patients in comparison to healthy controls. Moreover, patients with vascular arterial events were observed to have a significant increase in KIR2DS2and KIR2DL2gene frequencies when compared with patients without events. We also found that Chinese SLE patients had higher genotypic frequencies of KIR2DL2and KIR2DS1and were frequently observed with two more activating KIR genes than in controls. Stimulatory and inhibitory KIRs of SLE patients were found to be functional on lupus T cells as the DNA of these cells were hypomethylated. However, the mAbs of KIR2DL3/L2/S2and KIR2DL1/S1were used for flow cytometry as the stimulatory and inhibitory receptors could not be distinguished without genotyping nor were they related to any patient characteristics; thus, no conclusion was clearly established.ObjectiveTo investigate the expression pattern of Killer cell immunoglobulinlike receptors (KIR) on natural killer (NK)(CD3-CD56+) cells, natural killer T (NKT)(CD3+CD56+) cells, and subsets of T (CD3+CD56-) cells in SLE. To discussion the correlation between the expressions of CD158a/h, CD158b/i/j on NK-, NKT-, T-cells and SLEDAI score, immunological features of SLE patients. To investigate relative transcription_for KIR2DL1, KIR2DS1and KIR2DL2in NK-, NKT-and T-cells in SLE.Method1. Patients and controlsBoth SLE patients and controls were classified as2DS1+2DL1+groups (SLE patients:n=16; controls:n=12) and2DS2-2DL2+2DL3+groups (SLE patients:n=12; controls:n=8). The clinical laboratory of the hospital offered the immunological indices of SLE patients. Scatter Turbidimeter was used to measure the serum level of IgG, IgA, IgM, C3and C4; IIFT was used to measure ANA, and ELISA was used to measure ds-DNA, ss-DNA, AnuA, AHA, rRNP and SM. The cohort was genotyped for the presence or absence of KIR genes by using PCR-SSP.2. Flow cytometric analysisThe PBMC were isolated and were then analyzed by EPICS XL-4flow cytometry. Monoclonal antibodies as follows:(i) anti-human CD3(PE),(ii) anti-human CD56(Cy5),(iii) FITC-CD158a/h (EB6)(KIR2DL1/KIR2DS1) and (iv) FITC-CD158b/i/j (GL183)(KIR2DL2/KIR2DL3/KIR2DS2).3. We analyzed the correlation between the expressions of CD158a/h, CD158b/i/j on NK-, NKT-, T-cells and SLEDAI score, immunological features of SLE patients by using the Pearson correlation coefficient (r). A two-tailed p-value≤0.05was considered to be statistically significant.4. A CD56multi-sort kit along with CD3magnetic beads were used to isolate the NK-, NKT-and T cells from the PBMCs taken from of SLE patient and healthy control. And RT-PCR analysis was used for quantification of KIR2DL1,2DS1,2DL2, and the control18S gene by the ABI PRISM7500detection system.Results1. In SLE patients, the proportion of NK cells was significantly lower as compared to the controls (p<0.001). Moreover, a reduction in the proportion of NKT cells was observed in the peripheral blood of SLE patients as compared to the controls (p<0.001). However, in the T-cells, no significant difference was detected between the two groups.2. The expression of CD158a/h on NK cells in the SLE patients was significantly increased when compared to the healthy controls (p<0.001). However, the expression of CD158b/i/j on NK cells in the SLE patients was significantly decreased when compared to the healthy controls (p<0.001). And a significant increase of CD158b/i/j MFI (p=0.036) was seen in the SLE group.3. The expression of CD158a/h on NKT cells in the SLE patients was significantly increased when compared to the healthy controls (p<0.001). The expression of CD158b/i/j on NKT cells in the SLE patients was significantly decreased when compared to the healthy controls (p=0.002).4. Expression of CD158a/h on T-cells was also significantly increased in the SLE patients (p=0.026).5. The proportion of CD158a/h+NK cells positively correlated with the serum levels of anti-dsDNA titer in SLE patients (R=0.742, p=0.001).6. The proportion of CD158b1/b2/j+NKT cells in PBMCs and the serum levels of IgG showed a significant negative correlation in SLE patients (R=-0.591, p=0.043).7. The results showed the up regulation of transcription for KIR2DS1(p=0.025) and down regulation of KIR2DL1(p=0.049) in NK cells from SLE patients compared to the controls.8. The transcription of KIR2DS1was up regulated in NKT cells from SLE patients (p=0.041).Conclusion1. Our results verified that SLE patients have increased stimulatory and decreased inhibitory KIRs expression on NK cells. The increased expression of KIR2DS1on NK cells in SLE patients may decrease their cytotoxic effect against the target cells, such as auto-reactive T-cells, thus, resulting in an auto-antibody production (for example, Anti-dsDNA antibodies). So abnormal expression patterns of KIRs on NK cells of SLE patients may influence their differentiation and functional activity, and these may participate in the pathogenesis of SLE.2. The abnormal KIRs expression on the NKT cells maybe functional and contributing to SLE pathogenesis at least partly through influence serum IgG level.3. Stimulatory and inhibitory KIRs are functional on lupus T-cells, and the over expressive stimulatory KIRs may take part in the activation of auto-reactive T-cells in SLE patients.4. The proportion of CD158a/h+NK-, NKT-and T cells appeared to increase in the SLE patients group, while their MFI levels did not show any significant difference. Therefore, the increased KIR2DS1mRNA may be the result of a general expansion of2DS1+lymphocytes, but not a very high expression among the small NK or NKT cell subset. BackgroundReactive arthritis (ReA) is sterile arthritis triggered by bacterial gastrointenstinal or urogenital infections. Although the pathogenesis of ReA remains unclear, genetic factors seem to play an important role. Different killer cell immunoglobulin-like receptors (KIRs) and their corresponding specific histocompatibility leukocyte antigen-C (HLA-C) ligand genotypes have been implicated in susceptibility and resistance to infections and autoimmune diseases but have, thus far, not been investigated in ReA.MethodsThis study was conducted in138ReA patients (65females,73males); aged18to69years (mean,37years) and151randomly selected healthy control individuals matched for ethnicity, age and sex. These subjects were genotyped for KIR genes and HLA-C alleles by polymerase chain reaction with sequence-specific primers.Results The frequencies of inhibitory KIR2DL2and KIR2DL5were significantly lower in the ReA patients than in the controls (P=0.005and P=0.033, respectively). The presence of more than seven inhibitory KIR genes was protective (P=0.016). Moreover, we found that activating KIR2DS1alone or-in combination with the HLA-C1C1genotype (which indicates the absence of the HLA ligands for their homologous inhibitory receptor KIR2DL1) is associated with susceptibility to ReA (P=0.039and P=0.011, respectively), whereas KIR2DL2in combination with the HLA-C1ligand is associated with protection against ReA (P=0.039).ConclusionsThese observations indicate that high levels of activating and low levels of inhibitory KIR signals may affect the functions of NK cells and T cells. This imbalance enables the innate and adaptive immune responses of the host to be easily triggered by pathogens, resulting in the overproduction of local and systemic cytokines that contribute to the pathogenesis of ReA. BackgroundHypermethylated in cancer1(HIC1), which is located at17p13.3, a transcriptional repressor, resides in a CpG island that is hypermethylated in many types of human cancers and its targets genes involved in proliferation, tumour growth and angiogenesis. Now the Underhill group has generated a novel conditional Hicl knockout mice model. Preliminary analysis of these mice indicates that deficiency of this gene leads to a variety of skeletal malformations including, bone morphological anomalies, craniosynostosis and reduced bone mineral density,we found the salivary gland of the HIC1knockout mice embryos have developing disorder, so we try to figure out how does this happen and this would help us know better about HICl.And these studies will give us an opportunity to learn a new animal model for studying the etiology and treatment of salivary gland disorders.Methods1. Dissect different stages embryos out of the uterus in ice cold PBS and place in single wells containing PBS. SMG was isolated and taken images, genotyping of the embryos were done at the same time.2. Preparation of RNA and cDNA and quantitative real-time PCR, RNA was extracted from each pair of salivary glands using RNAeasy micro kit.The transcription levels of BTBD7, FGF10, MMP2, MMP9, Fnl (fibronectin), Inhibin beta A (Activin beta A), Inhibin beta B (Activin beta A) were tested.3. Whole-mount X-gal staining. Dissected E13.5SMGs were grown on filters overnight, prior to processing for X-gal staining..4. Immunohistochemistry and Immunofluorescence. Tissues were fixed in formalin overnight at4℃. Paraffin-embedded sections (7mm) were prepared for H&E staining, Immunofluorescence, and immunohistochemistry (IHC). We stained sections with the following primary antibodies:anti-Fnl (fibronectin), anti-colligenl, anti-Ki67, anti-AQP5. Secondary antibodies, conjugated to either the488Alexa-fluor or the594Alexa-fluor.Results1. HIC1(?)SMG consists primarily of undifferentiated epithelium composed of very few branches surrounded by undifferentiated, condensed mesenchyme.2. AQP5expression was reduced in HIC1(?)SMG.3. HIC1is located in mesenchyme of salivary gland4. Mesenchymal cells proliferation increased in HIC1(?)SMG.5. Fibronectin expressions were decreased in HIC1(?)SMG at later stages.6. Collagen I staining was decreased in order stages of HIC1(?)SMG.7. FGF-10transcripts were decreased in HIC1-/-SMG at E15.5.8. MMP2and MMP9transcripts were increased in HIC1(?)SMG at E14.59. Activin beta A and Activin beta B transcripts were increased in HIC1(?)SMG at stage E14.5while no Smad2staining difference can be found.Conclusions1. Impaired branching and size morphogenesis in HIC1(?)SMGs. Embryonic HIC1(?)SMGs are developmentally-delayed.2. As a functional marker of SMG, reduced AQP5expression means there is a function disorder in HIC1(?)SMGs. HIC1affects epithelial cell differentiation.3. HIC1affects the adjacent mesenchymal cells proliferation 4. HIC1maybe influence the inter lobe formation in the later stages of SMG by affecting Fibronectin’s expression.5. Collagen I,TGF10, MMP2and MMP9maybe also take part in HIC1signaling pathway in mice embryos salivary gland developing.更多还原