Hedgehog信号通路在脑胶质瘤的表达及其预后意义

目的 观测Hedgehog信号通路在脑胶质瘤的表达,探讨其表达在脑胶质瘤的预后意义.方法 选取118例原发性脑胶质瘤患者的手术切除标本,运用免疫组织化学方法检测Sonic hedgehog(Shh)、受体Patched(Ptch)及下游转录因子Gli1的表达,采用Kaplan-Meier生存分析和Cox比例风险回归模型评价脑胶质瘤患者的预后.结果 免疫染色结果显示Shh、Ptch和Gli1的阳性表达率随胶质瘤病理等级升高呈增强趋势(P＜0.01);随KPS评分的下降而成增强趋势(P＜0.01).生存分析表明,阳性表达Shh、Ptch和Gli1的胶质瘤患者总体存活率低于三者不表达的患者(P＜0.01).多因素Cox分析显示KPS(P＜0.05)、WHO grade(P＜0.01)、Shh(P＜0.05)、Ptch (P＜0.05)和Gli1(P＜0.05)是影响脑胶质瘤预后的独立因素.结论 脑胶质瘤的Shh-Ptch1-Gli1 信号通路处于激活状态,与脑胶质瘤的临床病理特征及预后参数密切相关,提示Hedgehog信号通路的活化在脑胶质瘤的恶性潜能和患者的生存时间起重要的预示作用.     

Hedgehog信号通路在人肝癌细胞株中的表达及意义

目的 探讨Hedgehog信号通路在人肝细胞癌细胞系中的表达及意义。 方法 采用半定量 RT-PCR 法检测PLC/PRF/5、HepG2及 SMMC-7721肝癌细胞株中Shh、Ptch1、Smo、Gli1、Gli2 mRNA 的表达;Western blot法检测PLC/PRF/5、HepG2及 SMMC-7721肝癌细胞株中Gli2蛋白的表达。 结果 除Shh外, Ptch1、Smo、Gli1、Gli2 mRNA在PLC/PRF/5、HepG2及 SMMC-7721细胞中均有不同程度表达。其中,Ptch1、Smo、Gli1、Gli2 mRNA在PLC/PRF/5及SMMC-7721细胞中的表达强度显著高于成人正常肝细胞,而Gli1 mRNA在HepG2细胞中的表达量与正常肝细胞无明显差异;Gli2蛋白在成人正常肝细胞中几乎没有表达,在HepG2细胞中的表达也与正常肝细胞无明显差别,而在PLC/PRF/5及 SMMC-7721细胞中,尤其是SMMC-7721细胞,Gli2的表达量异常增高。 结论 Hedgehog 信号通路的异常激活参与了肝细胞癌的发生发展过程,Gli2可能成为肝细胞癌重要生物学标志和生物治疗靶点。     

Dexamethasone induces osteogenesis via regulation of hedgehog signalling molecules in rat mesenchymal stem cells

Purpose

Hedgehog signalling plays an important role during the development of tissues and organs, including bone and limb. Dexamethasone (DEX), a synthetic and widely used glucocorticoid, affects osteogenesis of bone marrow mesenchymal stem cells (MSCs), while the signalling pathway by which DEX affects osteoblast differentiation remains obscure. This study aimed to investigate expressions of hedgehog signalling molecules Shh, Ihh and Gli1 during DEX-induced osteogenesis of rat MSCs in vitro.

Methods

DEX promoted osteoblast differentiation of MSCs at 10⁻⁸ mol/L from seven days to 21 days, demonstrated by enhancing alkaline phosphatase (ALP) activity and osteoblast-associated marker type I collagen expression during osteoblastic differentiation. Gene and protein expressions of hedgehog signalling molecules, Shh, Ihh and Gli1 were tested by RT-PCR and western blot analysis during osteoblast differentiation.

Results

Shh expression was increased compared to the control while Ihh and Gli1 expressions were decreased on both mRNA and protein level during DEX-induced osteoblast differentiation of MSCs from seven days to 21 days. Altogether, these data demonstrate that DEX can enhance Shh expression via a Gli1-independent mechanism during osteoblast differentiation of MSCs.

Conclusions

These results indicate that different patterns of hedgehog signalling are involved in DEX-induced osteogenesis and these findings provide insights into the mechanistic link between glucocorticoid-induced osteogenesis and hedgehog signalling pathway.     

Hedgehog信号传导通路相关基因在肝癌组织中的表达研究

目的 研究Hedgehog信号传导通路相关基因在肝癌组织中的表达,及其抑制剂对肿瘤细胞生长的影响.方法 免疫组化法检测14例肝癌组织切片、4个肝癌细胞系和正常肝细胞中Hedgehog信号传导通路成员Ihh、Ptch、Smo、Gli的蛋白表达;Western blot检测9例新鲜肝癌组织标本、6例正常肝组织标本和肝癌细胞系中Ihh、Ptch、Smo、Gh的蛋白表达;RT-PCR检测3肝癌组织标本和肝癌细胞系中Ihh、Ptch、Smo、Gli、Hip mRNA表达.结果 14例肝癌组织中免疫组化染色阳性的Gli 6例(42.9%),Ptch 10例(71.4%),Ihh 10例(71.4%),Smo 12例(85.7%).Western blot和RT-PCR检测显示,肿瘤组织中Gli基因的mRNA和蛋白表达均高于正常肝脏细胞,Hip基因的mRNA表达低于正常肝细胞.肝癌细胞系HepG2,Bel-7402和QGY-7701免疫细胞化学染色Pteh和Gli同时为阳性,证明为Hedgehog通路活化的细胞系.在环耙明(KAAD-cyclopamine)作用后,3个细胞系Ptch及Gli基因的mRNA表达下调(Ptch基因tHepG2=3.78,tBel-7402=9.03,tQGY-7701=5.63,Gli基因tHepG2=9.61,tBel-7402=4.15,tQGY-7701=20.30,均P＜0.05);QGY-7701组Hip基因RNA表达上调(t=4.70,P＜0.05).环耙明抑制肝癌细胞系QGY-7701的作用最明显.结论 原发性肝癌组织中Hedgehog信号传导通路有多种基因表达,环耙明有抑制肝癌细胞生长的作用.     

甲状腺乳头状癌组织中Shh和Gli1的表达及其临床意义研究

目的 通过检测Sonic Hedgehog信号通路关键分子Shh和Gil1在甲状腺乳头状癌表达的情况,探讨其与甲状腺乳头状癌临床病理特征的关系及临床意义.方法 应用免疫组织化学方法检测142例甲状腺乳头状癌患者癌组织及其癌旁组织病理蜡块标本中Shh和Gli1的表达情况,分析其与临床病理特征的关系.结果 Shh主要表达于...     

Hedgehog signaling pathway in neuroblastoma differentiation

Purpose

The hedgehog (Hh) signaling pathway is activated in some adult cancers. On the other hand, the Hh signaling pathway plays an important role in the development of the neural crest in embryos. The aim of this study is to show the activation of Hh signaling pathway in neuroblastoma (NB), a pediatric malignancy arising from neural crest cells, and to reveal the meaning of the Hh signaling pathway in NB development.

Methods

This study analyzed the expression of Sonic hedgehog (Shh), GLI1, and Patched 1 (Ptch1), transactivators of Hh signaling pathway, by immunohistochemistry in 82 NB and 10 ganglioneuroblastoma cases. All 92 cases were evaluated for the status of MYCN amplification.

Results

Of the 92 cases, 67 (73%) were positive for Shh, 62 cases (67%) were positive for GLI1, and 73 cases (79%) were positive for Ptch1. Only 2 (10%) of the 20 cases with MYCN amplification were positive for Shh and GLI1, and 4 cases (20%) were positive for Ptch1 (MYCN amplification vs no MYCN amplification, P ≦ .01). The percentage of GLI1-positive cells in the cases with INSS stage 1 without MYCN amplification was significantly higher than that with INSS stage 4. Of 72 cases without MYCN amplification, 60 were GLI1-positive. Twelve cases were GLI1-negative, and the prognosis of the GLI1-positive cases was significantly better than that of the GLI1-negative cases (P = .015).

Conclusions

Most of NBs without MYCN amplification were positive for Shh, GLI1, and Ptch1. In the cases without MYCN amplification, the high expression of GLI1 was significantly associated with early clinical stage and a good prognosis of the patients. In contrast to adult cancers, the activation of the Hh signaling pathway in NB may be associated with the differentiation of the NB.     

Laser irradiation promotes the proliferation of mouse pre-osteoblast cell line MC3T3-E1 through hedgehog signaling pathway

Low-level laser could promote osteoblast proliferation, and it has been applied in clinical practice to promote wound healing and tissue regeneration. However, the mechanism related to laser irradiation remains unclear. This study aimed to investigate the effects of low-level laser irradiation on the cell proliferation and the expressions of hedgehog signaling molecules Indian hedgehog (Ihh), Ptch, and Gli in vitro. In our present study, the MTT method was used to evaluate the effect on cell proliferation of laser irradiation on MC3T3-E1 cells. And cell cycle was examined by flow cytometry. Gene and protein expressions of hedgehog signaling molecules, including Ihh, Ptch, Smoothened (Smo), and Gli, were examined by qRT-PCR and western blot analysis. The results showed that laser irradiation at dosage of 3.75 J/cm² enhances the proliferation of MC3T3-E1 cells compared with control groups (p = 0.00). Moreover, laser irradiation (3.75 J/cm²) increased the cell amount at S phase (p = 0.00). In addition, the expressions of Ihh, Ptch, Smo, and Gli were significantly increased compared to the control during laser irradiation (3.75 J/cm²)-induced MC3T3-E1 osteoblast proliferation. After adding the hedgehog signaling inhibitor CY (cyclopamine), cell proliferation and Ihh, Ptch, Smo, and Gli expressions were inhibited (p = 0.00), and the cell amount at S phase was reduced compared with combination groups (p = 0.00). These results indicated that laser irradiation promotes proliferation of MC3T3-E1 cells through hedgehog signaling pathway. Our findings provide insights into the mechanistic link between laser irradiation-induced osteogenesis and hedgehog signaling pathway.     

Hedgehog signaling displays a biphasic expression pattern during intestinal injury and repair

Background/Purpose

Gastrointestinal injury is common clinically. The exact mechanism by which gastrointestinal repair occurs has yet to be well defined. Hedgehog (Hh) signaling is known to be involved in gastrointestinal development and repair of tissues such as skin and heart. The present study aimed to investigate the role of Hh in the repair of the small intestine.

Methods

i) To study acute intestinal injury, we optimized a mouse model of 5-flurouracil (5-FU) induced injury of the small intestine. Ileal tissues were evaluated for injury and repair markers at day 0, 2, 5, and 9. ii) Immunohistochemistry (Sonic hedgehog, Shh), in situ hybridization (Shh), and Ptch/LacZ transgenic mice were carried out to localize hedgehog expression. A33CrPr × ShhTg knock-in mice were bred to study the effect of Shh over-expression. qPCR of Shh, Ihh, Ptch, Bmp4 was carried out to quantify hedgehog signaling. iii) 5FU treated mice were then treated with a hedgehog inhibitor or saline (control) and the effects of Shh inhibition including apoptosis, proliferation, and mitosis were then compared.

Results

i) Immunohistochemistry and in situ hybridization of Shh, qPCR of hedgehog signaling pathway genes, and Ptch/LacZ staining results consistently showed down-regulation during the injury phase (P < 0.05) followed by up-regulation during the repair phase (P < 0.005). ii) Hh signaling inhibition following 5-FU induced injury augmented apoptotic activity (P < 0.05), suppressed mitotic activity (P < 0.005) in intestinal crypts, and reduced Paneth cell hyperplasia (P < 0.005). iii) Shh over-expression in conditionally knock-mice led to increased mitotic, Paneth, and goblet cells.

Conclusion

Hedgehog signaling pathway displays a biphasic expression pattern during the injury/repair of small intestine. It may play an important regulatory role in intestinal repair.     

FOXC2介导Hedgehog/Gli信号通路对乳腺癌侵袭及迁移的影响

目的探讨FOXC2介导Hedgehog/Gli信号通路通过调控上皮间质转化（EMT）途径参与乳腺癌侵袭及迁移的机制。 方法应用不同浓度环靶明（Cyclopamine）处理乳腺癌MDA-MB-231细胞,MTT法检测细胞增殖抑制率及计算药物半数抑制浓度（IC₅₀）;采用Cyclopamine或沉默FOXC2基因表达以阻断Hedgehog/Gli信号通路活化;通过Transwell小室体外侵袭实验及划痕实验分别检测阻断Hedgehog/Gli信号通路对MDA-MB-231细胞侵袭及迁移影响;Western blotting检测阻断前后Hedgehog/Gli信号通路分子Smoothened（Smo）、Gli1和EMT相关标志物FOXC2,E-cadherin及Vimentin蛋白表达变化。 结果与空白对照组比较,Cyclopamine可显著地抑制MDA-MB-231细胞增殖并呈现时效-量效关系,48 h的IC₅₀为25 μmol/L。Transwell小室体外侵袭实验及划痕实验均显示,与未转染组及Control-siRNA组相比,FOXC2-siRNA组和Cyclopamine组细胞穿膜数及迁移率均显著降低,差异有统计学意义（P<0.05）。Western blotting显示,与未转染组及Control-siRNA组相比较,FOXC2-siRNA组及Cyclopamine组Smo、Gli1及FOXC2蛋白表达显著降低,差异有统计学意义（P<0.05）。而沉默FOXC2表达可显著降低Vimentin蛋白表达及增加E-cadherin蛋白表达,差异有统计学意义（P<0.05）。 结论FOXC2通过介导Hedgehog/Gli信号通路从而调控EMT促进乳腺癌侵袭及迁移,提示阻断该信号通路有望成为乳腺癌靶向治疗新线索。     

Shh和Ptch在胆囊癌组织中的表达及其意义

目的 研究表明,Shh和Ptch是Hh传导通路的核心,消化系肿瘤中Hh通路的高度激活状态与消化系肿瘤的发生相关.本研究检测人胆囊腺癌标本中Shh和Ptch的表达,并探讨其意义.方法 采用免疫组化SP法对41例胆囊癌、21例胆囊腺瘤和20例正常胆囊组织中Shh和Ptch基因蛋白进行检测.结果 41例胆囊癌组织标本中Shh的阳性表达率为75.6%(31/41),Ptch的阳性表达率为78.0%(32/41).Shh和Ptch染色主要位于胆囊癌细胞的胞质和胞膜.20例正常胆囊组织中,Shh的阳性表达率为5%(1/20),Ptch的阳性表达率为10%(2/20).21例胆囊腺瘤组织中Shh的阳性表达率为4.7%(1/21),Ptch的阳性表达率为9.6%(2/21),各组间Shh和Ptch阳性表达率差异均有统计学意义(P＜0.001),而在不同性别、不同年龄组(年龄≥60岁及＜60岁)、不同病理组织学分级、Nevin不同分期、组织学不同分化程度、有无淋巴结或远处转移、有无伴发胆囊结石组间的Shh和Ptch阳性表达率表达差异均无统计学意义(P＞0.05).胆囊癌组织中Shh阳性表达率为75.6%与Ptch阳性表达率为78.0%之间存在显著正相关(r=0.72,P＜0.01).结论 原发性胆囊癌的发生可能与Hedgehog-Gli信号通路的异常活动密切相关.     

Indian Hedgehog信号通路在软骨细胞成熟及转分化过程中的调控作用

目的 探究Indian Hedgehog（IHH）信号通路对软骨内成骨过程中软骨细胞成熟以及转分化的影响。方法 取10日龄野生型小鼠的胫骨组织,采用原位杂交和免疫组织化学染色检测生长板区域IHH信号通路相关分子Ihh、Ptch1和Gli1的表达水平。构建肥大软骨细胞特异性Ihh基因敲除小鼠（Col10a1^Cre/+; Ihh^null/C）,并采用影像学检查和阿利新蓝染色评估该小鼠的骨骼发育状况。构建肥大软骨细胞IHH信号通路持续激活小鼠（Col10a1^Cre/+; R26Smo^M2/M2和 Col10a1^Cre/+; Ptch1^LacZ/C）,采用HE染色、原位杂交和TUNEL染色分别对受精15.5天胎鼠胫骨组织形态结构、Ihh（肥大软骨细胞分子标志物）和Col1a1（成骨细胞分子标志物）以及肥大软骨细胞凋亡水平进行检测;另外应用HE染色对10日龄小鼠的胫骨组织进行组织学分析。结果 肥大软骨细胞合成分泌IHH,但不表达Ptch1和Gli1。抑制肥大软骨细胞合成IHH蛋白会导致出生后小鼠出现侏儒症;X线检查结果显示小鼠出现严重的骨骼发育不良,包括胸廓狭小、球形头骨以及椎骨发育异常等表现。持续启动IHH信号通路时,胚胎早期软骨细胞成熟分化过程虽未见异常,但是出生后小鼠的骨小梁、骨内膜以及皮质骨等结构均出现一定的异常表现。结论 IHH信号通路虽然不参与肥大软骨细胞的终末分化过程,但在软骨细胞转分化的过程中起到了重要的调控作用。     

Stereotactic Radiosurgery for Human Glioma: Treatment Parameters and Outcome for Low vs. High Grade

Stereotactic radiosurgery (SRS) offers the precise, local delivery of radiation for the treatment of recurrent gliomas. We examined the comparative characteristics, treatments, and outcome in a population having with low– and high–grade gliomas. Between September 1991 and December 1995, 20 patients (13 males, 7 females) had SRS for low-grade [9 patients: World Health Organization (WHO) grade II] vs. high-grade (11 patients: 9 WHO grade IV and 2 WHO grade III) gliomas. The patients with low-grade gliomas were younger (mean age ± SE, 39.6 ± 5.4 years; range, 11.4–61.0 years) than those with high-grade gliomas (51.3 ± 13.9 years; range, 32.9–78.5 years) (P = 0.09). Tumor locations were similar in the two groups: lobar for 7 of 9 low-grade vs. 9 of 11 high-grade gliomas (P = NS) and diencephalic or cerebellar for the remainder. The initial surgical treatments were biopsy, subtotal resection, and total resection for three, three, and three patients with low-grade gliomas, vs. three, seven, and one patients with high-grade gliomas, respectively (P = NS). Except for three patients with low-grade gliomas, all patients had conventional postoperative fractionated external-beam radiotherapy. The doses were 5583 ± 342 vs. 5345 ± 261 cGy (P = NS) for low- vs. high-grade gliomas, respectively. Intervals from surgery and conventional radiation (if given) to progression and SRS tended to be longer for low-grade gliomas: 37.5 ± 9.5 vs. 30.6 ± 11.1 months (P = NS) for low- vs. high-grade gliomas, respectively. High-grade gliomas were larger. The diameters of the collimators that allowed enclosure of the enhancing tumor volume within the specified treatment isodoses were 22.4 ± 2.0 mm for low-grade vs. 29.8 ± 2.8 mm for high-grade gliomas (P = 0.02, ANOVA). SRS doses and isodose percentiles were similar, however, for the two groups: 1650 ± 191 cGy and 79 ± 4.0% vs. 1932 ± 182 cGy and75 ± 3.5% for low- vs. high-grade gliomas, respectively (P = NS, dose and isodose). All patients with high-grade gliomas were followed until death. The mean survival after SRS was 11.6 ± 1.5 months (42 ± 12 months after surgery). Five of nine patients with low-grade gliomas expired 31.6 ± 6.0 months after SRS (P < 0.001, Kaplan–Meier log rank) (74.0 ± 16.0 months after surgery). The four survivors have been followed for 8, 13, 35, and 38 months after SRS, respectively. Multivariate analysis shows that the category of histologic grade correlates significantly with survival after radiosurgery (P = 0.01). SRS may be an important therapeutic option for patients with recurrent gliomas, regardless of their grade.     

Hedgehog信号传导通路相关基因在肿瘤组织中的表达及其特异性抑制剂Cyclopamine对增殖、凋亡的影响

目的 观察Hedgehog信号传导通路相关基因在肿瘤组织中的表达,及其特异性抑制剂Cyclopamine对肿瘤细胞增殖、凋亡的影响.方法 免疫组织化学法检测80例人口腔鳞癌组织中Hedgehog信号传导通路成员Shh、Smo、Ptch、Gli-1表达;CCK8法检测对HSQ-89细胞增殖的影响;AnnexinV-PI双染流式细胞仪检测Cyclopamine对HSQ-89细胞凋亡的影响;逆转录-聚合酶链反应(RT-PCR)检测Cyclopamine处理后HSQ-89细胞凋亡相关蛋白表达变化.结果 Hh家族成员Shh、Smo、Ptch、Gli-1在80例口腔鳞癌中的表达率分别为:61.25%、56.25%、47.50%、53.75%,明显高于正常人;在低分化或发生淋巴结转移的病例中表达更高.Cyclopamine可抑制HSQ-89细胞增殖及诱导凋亡,且抑制效应呈浓度依赖性.Cyclopamine(0、5、10、20、40、80μmol/L)作用下,细胞增殖抑制率分别为0、3.0%、4.5%、28.2%、51.2%、62.7%,凋亡率分别为3.01%、3.36%、5.70%、9.42%.经Cyclopamine处理后HSQ-89细胞中bcl-2表达下降,bcl-xL、Bid等表达不变.结论 口腔鳞癌组织中Hedgehog信号转导通路有多种基因异常表达增高,Cyclopamine可通过抑制bcl-2表达而发挥抑制癌细胞增殖、诱导凋亡效应.     

Hedgehog信号通路转录因子Gli2表达对肝癌进展和生存预后的影响

目的探讨Hedgehog（Hh）信号通路转录因子Gli2在肝癌中的表达及其与预后的关系。 方法选取68例肝癌及20例肝血管瘤旁肝组织切除标本,采用免疫组织化学法检测Gli2蛋白表达,并分析Gli2表达与肝癌临床病理特征及预后的关系。 结果Gli2蛋白在肝癌组织中的表达率（63.2%）高于癌旁组织（11.8%）及正常肝组织（10.0%）(χ²=38.431、17528,均P<0.01);Gli2表达水平与肝癌分化程度、包膜是否完整、有无血管侵犯、早期复发及肝内转移相关（P<0.05）;Gli2蛋白高表达患者术后总生存率和无瘤生存率明显低于低表达者（P<0.05）;多因素分析表明,Gli2蛋白表达水平是影响肝癌患者术后总生存率及无瘤生存率的独立预后因素（HR=2.239,P<0.05;HR=1.917,P<0.05）。 结论Gli2的高表达与肝癌的侵袭转移特性相关,可作为评价肝癌患者术后预后的标志物之一。     

INDEPENDENTLY PREDICTIVE PROGNOSTIC VARIABLES AFTER RESECTION FOR COLORECTAL CARCINOMA

Background: Clinical variables such as surgical morbidity, comorbidity and follow-up have been claimed to influence ultimate survival in patients who have resection for colorectal cancer. It is unclear whether the effect of clinical covariates is confounding or independent. We have attempted to build a comprehensive model, which is capable of testing the dependence and importance of prognostic factors. Methods : A consecutive series of patients admitted between 1970 and 1988 and followed until 1992 had data recorded about presentation, pathology, hospitalization, aftercare and long-term outcome. The patients were also divided into two approximately equal groups that were cared for by one and seven surgeons, respectively. Clinical and pathological covariates were built into a Cox (multivariate) proportional hazard model of crude survival. This was achieved with the SPSSadvanced statistical package version 6.1. Comparison between groups was then performed of clinical and pathological factors and subsequent cancer management. Results : There were 207 patients whose average age was 75 years, median survival was 43 months and operative mortality was 4%. The Cox model was robust. Covariates that had independent survival effects were pathological stage (P= 0.0000), grade (P= 0.014), age (P= 0.018), heart disease (P= 0.001), and group (P= 0.0008). Some of the dependent variables were symptoms, type of surgery, complications and length of stay. The groups, however, were well matched for age, stage, substage and comorbidity. Furthermore there were no substantial differences in mortality, complications or follow-up frequency. There was a significant survival difference (P = 0.0003) between groups, which was restricted to patients who were in clinicopathological stages B and C. Within stages B and C there was a significant (P = 0.008) survival difference between patients who were or were not treated for recurrent disease. Diagnosis of recurrence was pursued more aggressively (P < 0.01), and decisions to treat recurrent disease were made more frequently in group 1 (P= 0.0002). Conclusions: Pathology, comorbidity and the management of recurrence all have a significant independent effect upon crude survival after colorectal carcinoma resection.