家蚕escargot基因的克隆及启动子活性鉴定

果蝇escargot(esg)基因作为表皮成组织细胞标记基因,可以指示果蝇成虫表皮形成过程。果蝇成虫雌较雄多1个体节,家蚕成虫与果蝇体节数相反。利用同源检索和RACE克隆获得果蝇esg在家蚕中的同源基因Bmesg,发现其ORF为1128 bp,共编码375 aa。进一步克隆该基因上游约2000 bp的调控序列esgp2k,细胞转染证明esgp2k有驱动活性。通过启动子截短及荧光素酶活性分析,证明起始密码子上游-270^-190 bp为esgp2k活性关键区域,并成功克隆长270 bp的核心启动子esgp7。上述结果为寻找家蚕成组织细胞潜在分子标记,进而研究家蚕雌雄体节数量差异的分子机制奠定了基础。

Expression of adhesion molecules on mature cholangiocytes in canal of Hering and bile ductules in wedge biopsy samples of primary biliary cirrhosis

AIM：To examine the expression of intercellular adhesion molecule-1 （ICAM-1） and lymphocyte function-associated antigen-1（LFA-1）expression on canals of Hering （COH）and bile ductules associated with the autoimmune process of bile duct destruction in primary biliary cirrhosis（PBC）．METHODS：Ten wedged liver biopsies of PBC（five cases each of stages 2 and 3）were studied. The liver specimens were processed for transmission electron microscopy. Immunohistochemistry was performed using anti-ICAM-1 and anti-LFA-1 mouse mAbs.In situ hybridization was done to examine the messenger RNA expression of ICAM-1 in formalin-fixed．paraffin-embedded sections using peptide nucleic acid probes and the catalyzed signal amplification （CSA）technique．Immunogold-silver staining for electron microscopy was Derrormed using anti-ICAM and anti-LFA-1 mouse mAbs．The immunogold particles on epithelial cells of bileductules and cholangiocytes of CoH cells were counted and analyzed semi-quantitatively．Western blotting was performed to confirm ICAM-1 protein expression．RESULTS：In liver tissues of PBC patients．immunohi-stochemistry showed aberrant ICAM-1 expression on the plasma membrane of epithelial cells lining bile ductules，and also on mature cholangiocytes but not on hepatocytes in CoH．LFA-1-positive lymphocytes were closely associated with epithelial cells in bile ductules．ICAM-1 expression at protein level was confirmed by Western blot．In situ hybridization demonstrated ICAM-1 mRNA expression in bile ductules and LFA-1 mRNA in lymphocytes infiltrating the bileductules．By immunoelectron microscopy，ICAM-1 was demonstrated on the basal suface of epithelial cells in bile ductules and on the luminal surfaces of cholangiocytes in damaged COH．Cells with intermediate morphology resembling progenitor cells in Coil were not labeled with ICAM-1 and LFA-1．CONCLUSION：De novo expression of ICAM-1 both on mature cholangiocytes in COH and epithelial cells in bile ductules in PBC implies that lymphocyte-induced destruction through adhesion by ICAM-1 and binding of LFA-1-expressing activated lymphocytes takes place not only in bile ductules but also in the COH．

Isolated pancreatic granulocytic sarcoma: A case report and review of the literature

Granulocytic sarcoma (GS) is an extramedullary tumor mass consisting of immature myeloid cells. Isolated pancreatic granulocyte sarcoma is extremely rare. We report a very unusual pancreatic granulocytic sarcoma in a patient without acute myeloid leukemia. The patient presented with acute epigastric pain because of splenic infarction due to a mass consisting of myeloblasts in the pancreatic tail. The patients underwent splenectomy and distal pancreatectomy. Pathology and immunohistochemistry suggested a GS. Despite local surgery, an isolated tumor recurred 2 mo after operation and the patient died 3 mo after removal of the tumor. Only 7 reported cases of pancreatic GS were identified in the literature and the mass was located in the pancreatic head. This is the first report of GS in the pancreatic tail with splenic infarction.

Significance and relationship between infiltrating inflammatory cell and tumor angiogenesis in hepatocellular carcinoma tissues

AIM: To investigate the relationship between infiltrating inflammatory cell and tumor angiogenesis in hepatocellular carcinoma (HCC) tissues and their clinicopathological features.METHODS: The paraffin-embedded specimens from 70 cases with HCC were stained using EliVision immunohistochemistry with mAbs against CD68, tryptase,and CD34. The counts of tumor-associated macrophage (TAM), mast cell (MC) and tumor microvessel (MV) were performed in the tissue sections.RESULTS: The mean counts of TAM, MC, and MV in HCC tissues were significantly higher than those in pericarcinomatous liver tissues (TAM: 69.31± 11.58 vs 40.23±10.36; MC: 16.74±5.67 vs 7.59±4.18; MV:70.11±12.45 vs 38.52± 11.16, P＜0.01). The MV count in the patients with metastasis was markedly higher than that with non-metastasis (P＜0.01). In addition, the MC count in the patients with poorly differentiated HCC was obviously higher than that with well differentiated HCC (P＜ 0.01). The correlation analysis showed that the TAM count was significantly correlated with the count of MV(r=0.712, P＜0.01), and the MC count was obviously correlated with the MV count (r= 0.336, P＜ 0.05).CONCLUSION: TAM and MC might be closely related to the enhancement of tumor angiogenesis. The MV count might be associated with tumor invasion and metastasis.Moreover, the MC count might be associated with tumor differentiation and prognosis of HCC.

Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

AIM： To identify the role of herbal compound 861 （Cpd 861） in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells （HSCs）. METHODS： mRNA levels of collagen types I and III, matrix metalloproteinase 1 （MMP-1）, matrix metalloproteinase 2 （MMP-2）, membrane type-1 matrix metalloproteinase （MT1-MMP）, tissue inhibitor of metalloproteinase 1 （TIMP-1）, and transforming growth factor β1 （TGF-β1） in cultured-activated HSCs treated with Cpd 861 or interferon-γ, （IFN-γ,） were determined by real-time PCR. RESULTS： Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-β1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to （6.3 ＋ 0.3）- fold compared to the control group. CONCLUSION： The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type Ⅲ and TGF-β1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

A Second Protein Marker of Caveolae:Caveolin-2

Caveolin-2,a protein about 20 kD,is a major component of the inner surface of caveolae,small invaginations of the plasma membrane.Similar with caveolin-1 and caveolin-3,it serves as a protein marker of caveolae.Caveolin-1 and-2 are located next to each other at 7q31.1 on human chromosome,the proteins encoded are co-localized and form a stable hetero-oligomeric complex,distributing similarly in tissue and cultured cells.Caveolin-3 is located on different chromosomes but confirmed to interact with caveolin-2.Caveolin-2 is similar to caveolin-1 in many respects but differs from the latter in functional domains,especially in G-protein binding domain and caveolin scaffolding domain.The mRNAs of both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during differentiation of 3T3-L1 cells to adipocytes.Caveolin-2-deficinet mice demonstrate clear pulmonary defects,with little or no change in caveolin-1 expression and caveolae formation,suggesting that caveolin-2 plays a selective role in lung functions.Caveolin-2 is also involved in lipid metabolism and human cancers.

Dynamic contrast-enhanced magnetic resonance perfusion weighted imaging in astrocytomas: correlation with histopathology and immunohistochemistry

Objective:To investigate magnetic resonance perfusion weighted imaging and its relationship with the grading and the expression of vascular endothelial growth factor (VEGF) and angiogenesis in astrocytomas. Methods: A collection of 34 patients with astrocytomas proved by surgery and pathology were examined by magnetic resonance imaging(MRI), with 26 cases of gradeⅠ-Ⅱ(low-grade) and 8 cases of grade Ⅲ-Ⅳ(high-grade). MR perfusion images were obtained with spin-echo echo planar imaging (SE-EPI) techniques. Expression of VEGF was examined by immunohistochemical method of streptavidin-biotin-peroxidase(SP). The vascular development was measured by micro-vascular density (MVD) which was immunostained with anti-factor Ⅷ-related antigen monoclonal antibody. Results: Both of the expression of VEGF and the angiogenesis in 34 cases of astrocytomas were significantly correlated to the maximum relative cerebral blood volume (Max rCBV) (r=0.604, P<0.001;r=0.625, P<0.001, respectively). The Max rCBV and the expression of VEGF, MVD in high-grade astrocytomas were significantly higher than that of in low-grade astrocytomas (t= 3.0, P=0.017; t=7.08, P=0.01;t=3.37,P=(0.011,) respectively). Conclusion: MR perfusion weighted imaging might be a valuable method in in vivo study of the angiogenesis of astrocytomas and evaluating their malignant degree and prognosis.

Study on biocompatibility of PDLLA/HA/DBM with co-cultured human osteoblasts in vitro

Objective: To evaluate the osteocompatibility of D, L-polylactic/hydroxyapatite/decalcifying bone matrix (PDLLA/HA/DBM), and compare with PDLLA and DBM. Methods: Human primary osteoblasts isolated from the femoral head of patients were inoculated onto PDLLA/HA/DBM, PLA and DBM respectively. The proliferation rate and collagen Ⅰ expression were detected. The interface between biomaterial and osteoblasts was investigated with phase contrast microscopy and electron scanning microscopy. Results: Best proliferation rate was observed with the PDLLA/HA/DBM and followed by DBM and PLA, suggesting that PDLLA/HA/DBM satisfying most requirements for the cultivation of human osteoblasts. Scanning electron microscopy showed the morphology of osteoblasts was correlated with the proliferation data. The cells, well spread and flattened, were attached closely on the surface of biomaterial with an arched structure and had normal morphology. The extracellular collagenous matrixs covered the surface of biomaterial and packed the granules of biomaterial. Conclusion: PDLLA/HA/DBM can form osteointerface early and have a good biocompability.

Influence of different gravitational environments on the mechanotrans-duction in osteoblasts

Objective: To observe the effects of different gravitational environments on release of prostaglandin E2 in rat calvarial osteoblasts induced by fluid shear stress (FSS) so that to investigate the influence of different gravity on mechanotransduction in osteoblasts. Methods: Osteoblasts were isolated from neonatal rat calvariae and then were set to three groups. Each was cultured in one gravitational environment; 1G terrestrial gravitational environment (control), simulated weightlessness achieved by using clinostat and 3G gravitational environment achieved by using centrifuge for 60 h, then osteoblasts were treated with 0. 5 Pa or 1. 5 Pa FSS in a flow chamber for 1 h. The release of PGE2 in osteoblasts was determined. Results: In 1G gravitational environment, the release of PGE2 was significantly increased along with the sustaining of FSS treatments (P<0. 01), but there was no remarkable difference between the responses to 0. 5 Pa FSS and 1. 5 Pa FSS (P>0. 05). While in simulated weightlessness environment group, no detectable release of PGE2 was found with the treatment of 0. 5 Pa FSS (P<0. 01), and the release of PGE2 was delayed and the amount of PGE2 production was remarkably decreased with 1. 5 Pa FSS treatment as compared with that of 1G group (P<0. 01). The responsiveness of osteoblasts cultured in 3G gravitational environment to FSS was similar to that of 1G group. Conclusion: These results indicate that in vitro the mechanotransduction in osteoblasts iss affected by stimulated weightlessness, whereas it is not altered in 3G gravitational environment.

Mechanistic Insights of Cells in Porous Scaffolds via Integrated Culture Technologies

This research aimed to combine 3 cell and tissue culture technologies to obtain mechanistic insights of cells in porous scaffolds. When cultivated on 2D （2-dimensional） surfaces, HDFs （human dermal fibroblasts） behaved individually and had no strict requirement on seeding density for proliferation; while HaCat cells relied heavily on initial densities for proliferation and colony formation, which was facilitated when co-cultured with HDFs. Experiments using a 3D CCIS （3-dimensional cell culture and imaging system） indicated that HDFs colonised openpores of varying sizes （125-420 ~tm） on modular substrates via bridge structures; while HaCat cells formed aperture structures and only colonised small pores （125 txm）. When co-cultured, HDFs not only facilitated HaCat attachment on the substrates, but also coordinated with HaCat cells to colonise open pores of varying sizes via bridge and aperture structures. Based on these observations, a 2-stage strategy for the culture of HDFs and HaCat cells on porous scaffolds was proposed and applied successfully on a cellulosic scaffold. This research demonstrated that cell colonisation in scaffolds was dependent on multiple factors; while the integrated 2D＆3D culture technologies and the 3D CCIS was an effective and efficient approach to obtain mechanistic insights of their influences on tissue regeneration.

Expression of VEGF-C and b-FGF in Lung Cancer and Its Relationship with Cancer Progress

Objective： to observe expression of vascular endothelial growth factor-C （VEGF-C） and alkaline fibroblast growth factor （b-FGF） in tissues of lung cancer, and its relationship with cancer metastasis.Methods： to adopt immunohistochemical methods, analysis of 60 cases of lung tissue expression of VEGF-C and b-FGF in the situation.Result： positive rates of VEGF-C and b-FGF in lung cancer are respectively 56.67% and 63.33%; expression of VEGF-C and b-FGF in lung cancer is not related to pathological grades, pathologic stages or ages of patients （P 〉 0.05）,but closely related to TNM stages and existence of lymph node metastasis （P 〈 0.01）. IMVD in center of lung cancer tissues is obviously higher than surrounding area, with significant differences （P 〈 0.01）. Conclusion： expression of VEGF-C and b-FGF is related to lung cancer progress.

关于“氧疗”的对话(上)

林大夫:主治医师 张大夫:实习医师 张:林大夫,为什么前几年满街的“氧吧”、“氧室”很快减少或消失了呢? 林:这实际上反映了人们对氧疗的认识由误解到正确的过程。氧在大气中占21％,正常情况下。

低能运动益于健康长寿

不久前,美国圣路易斯华盛顿大学运动医学研究中心的专家们指出,与以往的健身观念大相径庭的是,有益于人类健康长寿的锻炼不是那些高难度、大负荷量、剧烈的运动,而是那些普通项目、灵活轻松,不拘形式、强度较小、耗能较低的低能运动(即每周从运动中消耗1800—2200卡左右的热量或运动时的代谢量为4—5.5MET·梅脱)的运动。哈佛医学院预防医学研究所的坎内斯博士对此论证表示肯定。坎内斯与他的同事曾对8000余名中老年人进行了为期12年的跟踪调查,结果表明,长期参加低能运动的人比不参加任何运动或偶尔进行剧烈运动的人死亡率降低2.5倍,罹患脑心血管病、糖尿病、癌症、早老性痴呆的发病率减少35％左右,其寿命延长4——10年。

Monocyte-secreted Wnt5a interacts with FZD5 in microvascular endothelial cells and induces angiogenesis through tissue factor signaling

Angiogenesis during reactive and pathologic processes is characteristically associated with inflammation. Inflammatory cells partici- pate in angiogenesis by secreting different molecules that affect endothelial celt functions. We had previously shown that induced tissue factor （TF） expression in activated rnicrovascular endothelial celts （rn EC） is able to induce angiogenesis via autocrine regulation. However, the signals that induce TF expression in mEC are not fully known. Here, we demonstrate that rnonocyte paracrine cross-talk with mECs triggers rnEC-TF expression. We have identified that rnonocyte-secreted Wnt5a induces TF expression in rnEC and function-ally induces celt rnonolayer repair and angiotube formation in vitro as well as rnicrovesset formation in vivo. Monocyte-secreted Wnt5a activates FZD5 in mECs, which signals to induce the release of intraceUular Ca2＋ and increase NFKB transcription activity and TF gene expression. In sum, WntSa secreted by monocytes signals through the noncanonical Wnt-FZD5 pathway in mECs to induce TF expression that induces angiogenesis by autocrine regulation.

Eplerenone inhibits atrial fibrosis in mutant TGF-β1 transgenic mices..

The purpose of the present study was to study the impacts of eplerenone （EPL）, an antagonist of mineralocorticoid receptors （MR）, on atrial fibrosis in a mouse model with selective fibrosis in the atrium, and to explore the possible mechanisms. Using mutant TGF-β1 transgenic （Tx） mice, we first demonstrated that EPL inhibited atrial fibrosis specifically and decreased mac- rophage accumulation in the atria of these mice. Results from immunohistochemistry and western blotting showed that EPL attenuated protein expression of fibrosis-related molecules such as connective tissue growth factor （CTGF） and fibronectin in the atria of Tx mice. In culture, EPL inhibited gene expression of fibrosis-related molecules such as fibronectin, ct-SMA, and CTGF in TGF-β1-stimulated atrial fibroblasts, Finally, using a co-culture system, we showed that TGF-β1 stimulated atrial fi- broblasts induced migration of macrophages and this was blocked by EPL. EPL also blocked TGF-β1 induced gene expression of intedeukin-6 （IL-6） in atrial fibroblasts. Therefore, we conclude that EPL attenuated atrial fibrosis and macrophage infiltra- tion in Tx mice. TGF-I31 and IL-6 were involved in the impacts of EPL on activation of atrial fibroblasts and interactions be- tween fibroblasts and macrophages.

The role of the microenvironment on the fate of adult stem cells

Adult stem cells(SCs) exist in all tissues that promote tissue growth, regeneration, and healing throughout life. The SC niche in which they reside provides signals that direct them to proliferate, differentiate, or remain dormant; these factors include neighboring cells, the extracellular matrix, soluble molecules, and physical stimuli. In disease and aging states, stable or transitory changes in the microenvironment can directly cause SC activation or inhibition in tissue healing as well as functional regulation. Here, we discuss the microenvironmental regulation of the behavior of SC and focus on plasticity approaches by which various environmental factors can enhance the function of SCs and more effectively direct the fate of SCs.

Effect of puerarin on matrix metalloproteinase-2 in human fetal scleral fibroblasts treated with low frequency electromagnetic fields

OBJECTIVE:To study the effect of puerarin on matrix metalloproteinase-2(MMP-2)gene and protein expression in human fetal scleral fibroblasts(HFSFs)exposed to extremely low frequency electromagnetic fields(ELF-EMF).METHODS:Cultured HFSFs were exposed to 0.2mT ELF-EMF for 24 h.The experimental groups were divided into subgroups treated with 0,0.1,1and 10μM puerarin respectively.The expression of MMP-2 mRNA and protein were detected with real-time polymerase chain reaction and western-blot analysis respectively.RESULTS:MMP-2 mRNA and protein expression increased by 0.793 and 1.130 folds respectively under the exposure of ELF-EMFs at 0.2 mT flux density for24 h.Puerarin at the concentration of 0.1μM reversed this effect by 8.53%in mRNA and by 17.97%in protein expression(P<0.05).The effect was more prominent at higher concentrations(1 and 10μM,P<0.01).CONCLUSION:Exposure to ELF-EMFs increased the expression of MMP-2 mRNA and protein in HFSF cells.Puerarin reversed the action to some extent in a specific concentration range.Our results implied that the puerarin might protect scleral tissue from increased expression induced by exposure to ELF-EMFs.

Expression of hunchback during oogenesis and embryogenesis in Locusta migratoria manilensis(Meyen)

hb(hunchback) is a contributing factor in anteroposterior axial patterning of insects.Although the hb function in Locusta migratoria manilensis has been investigated,its expression pattern remains unknown.Here,the mouse polyclonal antibody was produced against Hb fusion protein,and then its expression pattern during oogenesis and embryogenesis of L.migratoria manilensis was examined by immunohistochemical staining.Hb protein was detected in the oocyte nucleus which was positioned centrally within the developing oocyte.The oocyte nucleus gradually moved to the posterior end of the egg along with the oocyte maturing.In freshly laid eggs,Hb formed gradient at the posterior end of the egg,and then hb was expressed as a band in the middle of the blastodisc.As the blastodisc differentiated into the head and trunk,the expression region became wide,which would develop into spatial gnathal and thoracic segments.With abdominal segmentation,the expression domain in the gnathal and thoracic region became faint and eventually faded out,while the Hb expression domain appeared at the posterior growth zone in a discontinuous expression manner.The hb expression pattern of L.migratoria manilensis is greatly similar to that of other locusts,such as Schistocerca americana and another L.migratoria.Compared with other insects,hb expression is conserved in the gnathal and thoracic domains,while divergent in oogenesis and abdomen.

Regulatory Action of Charred Gossamer Urocteae on the Functions of Mouse Oral Fibroblasts

Objective： To explore the influence of charred Gossamer urocteae （CGU） on the functions of primary cultured mouse oral fibroblasts and reveal its mechanism in wound healing. Methods： CGU was extracted with different solvents and ethanol extract （EE）, ethyl acetate fraction （EF）, n-butanol fraction （BF） and aqueous fraction （AF） were obtained. The effects of different fractions on the proliferation, matrix metaUoproteinase-2,9 （MMP-2,9） activities, synthesis of collagen and tissue inhibitor of metalloproteinase 1 （TIMP-1） in the mouse oral fibroblasts were determined by MTT, gelatin zymography, chloramine-T method, and enzyme-linked immunosorbent assay （ELISA） respectively. Results： EE, EF and BF at high concentrations could significantly inhibit proliferation of fibroblasts （P〈0.05 or P〈0.01）, and at low concentrations EF and BF could promote proliferation of fibroblasts, and BF and AF could significantly inhibit collagen synthesis （P〈0.05 or P〈0.01）. EE, EF and AF at high concentrations could significantly increase the MMP-9 activity, and BF and AF could significantly inhibit synthesis of TIMP-1. Conclusion： CGU at high concentrations can inhibit the proliferations of fibroblasts and synthesis of collagen, and in healing of wound, CGU at high concentrations possibly has the functions of anti-fibrosis and anti-scar, and the mechanism to promote degradation of collagen is possibly related to the increase in MMP-9 activity and the inhibition of TIMP-1 synthesis.