短期进入高原从事高强度工作所致高原反应是值得探讨的问题,查阅文献,探讨其病因及发病机理、临床表现,总结国内外在诊断、预防及治疗方面的经验,探索一套可行、有效的预防及治疗措施,具有重要的临床意义。
目的 探讨HIF-1α和BAK蛋白在胃癌中的表达情况，以及二者在胃癌中的相互关系及作用。方法 应用免疫组化SABC染色法检测80例胃癌组织和20例正常胃组织中的HIF-1α和BAK蛋白的表达情况。结果 胃癌中HIF-lα和BAK蛋白的表达阳性率分别为56.3%（45/80）和67.5%（54/80），而在胃正常组织中分别为5.0%（1/20）和20.0%（4/20），二者在胃癌中的表达显著高于胃正常组织，其差异有统计学意义（P＜0.05）。HIF-1α蛋白表达与胃癌组织的浸润范围、分化程度及淋巴结转移有关（P＜0.05），与临床分期、年龄及性别无关（P＞0.05）；BAK蛋白表达与胃癌浸润及分化程度相关（P＜0.05），与淋巴结转移、临床分期、年龄及性别无关（P＞0.05）。胃癌组织中HIF-1α与BAK蛋白的阳性表达之间呈正相关（列联系数r=0.056，P＜0.05）。结论 HIF-1α与BAK蛋白在胃癌的临床分期及浸润转移中存在关系，这对于研究胃癌的发生和发展，以及对于探索以二者为靶点的抗肿瘤治疗有重要意义。
ObjectiveTo elucidate whether hypoxia induced factor-1α (HIF-1α) gene improved hypoxia tolerant capability of bone marrow mesenchymal stem cells uptake(MSCs) or not and whether the capability was related to glucose uptake increase in hypoxia MSCs ex vivo or not. MethodsMSCs were randomly divided into normoxia non-HIF-1α transfection group (control group), normoxia HIF-1α transfection group, hypoxia non-HIF-1α transfection group, and hypoxia HIF-1α transfection group and then each group was cultured with normoxia (5% CO2 at 37 ℃) or hypoxia (94% N2, 1% O2, 5% CO2 at 37 ℃) for 8 h, respectively. Finally, the expressions of HIF-1α were detected by immunocytochemistry, RT-PCR, and Western blot methods, respectively. Apoptosis ratio (AR) and death ratio (DR) were tested by flow cytometry. The proliferation was detected by MTT method. Glucose uptake was assayed by radiation isotope method. Results① Compared with the normoxia non-HIF-1α transfection group, the expression of HIF-1α mRNA significantly increased (Plt;0.01) in the normoxia HIF-1α transfection group except for its protein (P=0.187); Both of mRNA and protein expressions of HIF-1α in the hypoxia HIF-1α transfection group were significantly higher than those in the hypoxia non-HIF-1α transfection group (Plt;0.01). ② The AR (P=0.001) and DR (P=0.003) in the hypoxia HIF-1α transfection group were significantly lower thanthose in the hypoxia non-HIF-1α transfection group, both of which were significantly higher than those in the normoxia non-HIF-1α transfection group (Plt;0.01). ③ The proliferation of MSCs in the hypoxia HIF-1α transfection group was significantly higher than that in the hypoxia non-HIF-1α transfection group (P=0.004), which significantly lower than that in the normoxia non-HIF-1α transfection group (P=0.001). ④ Compared with the hypoxia non-HIF-1α transfection group, the 3H-G uptake capability (P=0.004) of MSCs significantly increased in the hypoxia HIF-1α transfection group, which was significantly lower than that in the normoxia non-HIF-1α transfection group (P=0.001). ⑤ There were significantly negative relation between AR and HIF-1α protein (r=－0.71，P=0.005) or 3H-G uptake (r=－0.65，P=0.004), and significantly positive relation between HIF-1α protein expression and 3H-G uptake (r=0.77, P=0.003). ConclusionHIF-1α gene significantly improves anti-hypoxia capability of MSCs, which is fulfilled by increasing glucose upake.
Objective To investigate relationship between hypoxia microenvironment and occurrence and development of hepatocellular carcinoma (HCC). Method The relevant literatures on researches of the relationship between the hypoxic microenvironment and the HCC were review and analyzed. Results The hypoxia microenvironment played an important role in inducing the drug resistance and angiogenesis of the HCC cells, and it was an important factor of affecting the ability of tumor metabolism, invasion, and migration. The hypoxia microenvironment could up-regulate the expression of hypoxia-inducible factors (HIFs) and promote its transcriptional activity, promote the expression of the vascular endothelial growth factor gene, and regulate the neovascularization in the tumor. Among them, the HIF-1α played a major role in regulating the angiogenesis, immune escape, tumor invasion and metastasis-related gene expression, participating in the glycolysis, regulating lysyl oxidase 2 and thus regulated epithelial-mesenchymal transition, then promoted the invasion and metastasis of the HCC; HIF-2α was a key regulator of the malignant phenotype involving in the cell proliferation, angiogenesis, apoptosis, metabolism, metastasis, and resistance to chemotherapy. The hypoxia microenvironment posed some difficulties for the treatment of HCC, but it was also a potential therapeutic breakthrough. Conclusion Hypoxia microenvironment can promote invasion and metastasis of HCC through various mechanisms, which provides new targets and strategies for clinical treatment of HCC.
Objective To observe the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-β1 expression and to provide theoretical basis for deci phering the molecular mechanism of cl inical distraction osteogenesis. Methods The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining,the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-β1 at 0, 3, 6, 9, 12 and 24 hours after culture. Results The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing l ine was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outl ine was unclear, and at the cell compact district, massive mascul ine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that mascul ine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after15 days of culture. After al izarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after peration, and then dropped to the normal level. At various time points, TGF-β1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was sl ightly ber than that in the VEGF control group. In the experimental group, TGF-β1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time. Conclusion The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-β1.
【 Abstract 】 Objective To observe the effect of disruption of hypoxia inducible factor-1 α (HIF-1 α ) pathway by small hairpin RNA (shRNA) on chemosensitivity of human hepatocellular carcinoma (HCC) cells and to reveal the correlative mechanisms. Methods Plasmid of pshRNA-HIF-1α was transfected into HepG2 cells by lipofectamine. HepG2/pshRNA-HIF-1α (HepG2/pshRNA) cell lines were obtained by selection of HepG2 cells in G418. Meanwhile, plasmid of empty vector (pHK) was transfected as a control (HepG2/pHK). The mRNA and protein expression levels of HIF-1α and mdr1 were investigated by RT-PCR and Western blot respectively. Using CoCl2 to simulate the hypoxia condition, growth inhibition and apoptosis rates of HepG2 cells under different dosages of chemotherapeutic agents (adriamycin) were measured by MTT assay and flow cytometry (FCM) ． ResultsCompared with HepG2/pHK cells, the mRNA and protein expression levels of HIF-1αand mdr1 were obviously down-regulated in HepG2/pshRNA cells. At the same time, the proliferation inhibition and apoptosis rates were evidently increased after transfection with pshRNA-HIF-1α(P＜0.05)，which decreased the expression of HIF-1αto 82.18% at mRNA level and 75.51% at protein level. There was no significant effect of transfection pHK (Pgt;0.05). Conclusion These data demonstrates that HIF-1α interference by shRNA increased the sensitivity of HCC chemotherapy and the reversal of multidrug resistance, which may be done by down-regulating the transcription of mdr1 and the translation of P-gp. Blocking HIF-1αin HCC cells may offer an new avenue for gene therapy．
Autophagy is a lysosome dependent, conservative material degradation process, which exists in all eukaryotic cells and plays import roles in many pathophysiology process. Erectile dysfunction (ED) is a common male disease with multiple etiology. In recent years, more and more evidences have demonstrated that autophagy has a close relation to ED, therefore, we combine previous study to classify ED by hypoxia, aging, diabetes and other causes, and review the advances of autophagy in ED.
目的 研究缺氧诱导因子-1α（HIF-1α）与基质金属蛋白酶-2（MMP-2）在低分化胃癌中的表达，并探讨两者在低分化胃癌浸润、转移过程中的相互关系及作用。方法 采用SP免疫组化方法，检测54例低分化胃癌及20例正常胃组织石蜡标本中HIF-1α和MMP-2蛋白表达水平。结果 低分化胃癌组织中HIF-1α和MMP-2蛋白表达阳性率明显高于正常胃组织（Plt;0.05）。两者的表达与肿瘤的浸润深度、淋巴转移和TNM分期均有关（Plt;0.05），HIF-1α与MMP-2的表达呈正相关（r=0.580, Plt;0.05）。结论 HIF-1α和MMP-2在低分化胃癌的侵袭、转移过程中存在一定的协同作用，两者可能成为判断胃癌预后的指标。
ObjectiveTo build a lentiviral expression vector regulated by two targets 5 copies of HREs and hTERTp, express the target gene CDX2, and to test the activity of hTERT promoter by using LoVo cells for transfection. MethodsAfter the primer sets were designed, the hTERT promoter was cloned by PCR amplification from the genome of colon cancer. The CEA promoter was removed from the original vector pLEGFP-5HRE-CEAp by double digestion and PCR method, and then the hTERTp was introduced into the vector to construct the recombinant plasmid pLEGFP-5HRE-hTERTp. 5HRE-hTERTp was obtained by PCR, while the CMV promoter was removed from the original vector pLVX-EGFP-3FLAG by double digestion and PCR method, and then the 5HRE-hTERTp was introduced into the vector to construct the recombinant plasmid pLVX-5HRE-hTERTp-EGFP-3FLAG. The CDX2 was cloned by PCR amplification from GV230-CDX2-EGFP, and the EGFP was removed from the vector pLVX-5HRE-hTERTp-EGFP-3FLAG by double digestion, and then the CDX2 was introduced into the vector to construct the recombinant plasmid pLVX-5HRE-hTERTp-CDX2-3FLAG. LoVo cells ex vivo was transiently transfected by pLVX-5HRE-hTERTp-EGFP-3FLAG to evaluate the activity of hTERTp by detecting the expression of green fluorescence protein EGFP. ResultsPCR and sequencing analyzing showed that pLEGFP-5HRE-hTERTp, pLVX-5HRE-hTERTp-EGFP-3FLAG, and pLVX-5HRE-hTERTp-CDX2-3FLAG were sequenced correctly and the same as our designed. pLVX-5HRE-hTERTp-EGFP-3FLAG was successfully transfected into LoVo cells ex vivo and expressed green fluorescence protein EGFP, which showed that hTERTp was activated and promoted the expression of downstream gene. ConclusionThe lentiviral expression vector, pLVX-5HREhTERTp-EGFP-3FLAG and pLVX-5HRE-hTERTp-CDX2-3FLAG are successfully constructed, which lays the foundation of further research. But the function of dual-target regulation needs further proof.