PURPOSE:Effective therapy for visual loss caused by optic nerve injury or diseases has not been achieved even though the optic nerve has the regeneration potential after injury. This study was designed to modify amniotic epithelial cells (AECs) with basic fibroblast growth factor (bFGF) gene, preliminarily investigating its effect on transected optic nerve. METHODS:A human bFGF gene segment was delivered into rat AECs (AECs/hbFGF) by lentiviral vector, and the gene expression was examined by RT-PCR and ELISA. The AECs/hbFGF and untransfected rat AECs were transplanted into the transected site of the rat optic nerve. At 28 days post transplantation, the survival and migration of the transplanted cells was observed by tracking labeled cells; meanwhile retinal ganglion cells (RGCs) were observed and counted by employing biotin dextran amine (BDA) and Nissl staining. Furthermore, the expression of growth associated protein 43 (GAP-43) within the injury site was examined with immunohistochemical staining. RESULTS:The AECs/hbFGF was proven to express bFGF gene and secrete bFGF peptide. Both AECs/hbFGF and AECs could survive and migrate after transplantation. RGCs counting implicated that RGCs numbers of the cell transplantation groups were significantly higher than that of the control group, and the AECs/hbFGF group was significantly higher than that of the AECs group. Moreover GAP-43 integral optical density value in the control group was significantly lower than that of the cell transplantation groups, and the value in the AECs/hbFGF group was significantly higher than that of the AECs group. CONCLUSIONS:AECs modified with bFGF could reduce RGCs loss and promote expression of GAP-43 in the rat optic nerve transected model, facilitating the process of neural restoration following injury.

Background: Atherosclerotic renal artery stenosis (ARAS) and coronary artery disease (CAD) commonly co-exist. Some patients with unidentified ARAS may undergo cardiac surgery. While acute kidney injury (AKI) is a frequent and serious complication of cardiac surgery, we aim to evaluate the influence of ARAS on the occurrence of postoperative AKI in patients with normal or near-normal baseline renal function following cardiac surgery.Methods: A total of 212 consecutive patients undergoing aortography after coronary angiography and cardiac surgery were retrospectively studied for their preoperative and intraoperative conditions. AKI was defined as an absolute increase in serum creatinine of more than or equal to 0.3 mg/dl (>= 26.4 mu mol/l) or a percentage increase in creatinine of more than or equal to 50% (1.5-fold from baseline) after cardiac surgery. A propensity score-adjusted logistic regression models was used in estimating the effect of ARAS on the risk of postoperative AKI.Results: ARAS (>= 50%) was observed in 50 (23.6%) patients, and 83 (39.2%) developed AKI after cardiac surgery. A correlation existed between renal artery patency and preoperative-to-postoperative %Delta Cr in patients with ARAS (r = 0.297, P<0.0001). The propensity score-adjusted regression model showed the occurrence of postoperative AKI in patients with ARAS was significantly higher than those without ARAS (OR 2.858, 95% CI 1.260-6.480, P = 0.011).Conclusion: ARAS is associated with postoperative AKI in patients with normal or near-normal baseline renal function after cardiac surgery.

Despite advances in antibiotic therapy and intensive care, the mortality caused by systemic inflammatory response syndrome and severe sepsis remains high. The use of anti-inflammatory agents to attenuate inflammatory response during acute systemic inflammatory reactions may improve survival rates. Here we show that a newly synthesized 2-pyridone compound (FJU-C4) can suppress the expression of late inflammatory mediators such as iNOS and COX-2 in murine macrophages. The pro-inflammatory cytokines, including TNFα, IL-1β, and IL-6, were dose-dependently suppressed by FJU-C4 both in mRNA and protein levels. In addition, the expression of TNFα was inhibited from as early as 2 hours after exposure to LPS stimulation. The production of mature pro-inflammatory cytokines was also suppressed by pretreatment with FJU-C4 in either cell culture medium or mice serum when stimulated by LPS. FJU-C4 prolongs mouse survival and prevents mouse death from LPS-induced systemic inflammation when the dose of FJU-C4 is over 5 mg/kg. The activities of ERK, JNK, and p38MAPK were induced by LPS stimulation on murine macrophage cell line, but only p38MAPK signaling was dramatically suppressed by pretreatment with the FJU-C4 compound in a dose-dependent manner. NF-κB activation also was suppressed by FJU-C4 compound. These findings suggest that the FJU-C4 compound may act as a promising therapeutic agent against inflammatory diseases by inhibiting the p38MAPK and NF-κB signaling pathway.

Some bicyclic imides and triazole compounds have separately shown biological activities. We now combine these two structural features into the synthesis of triazolyl-substituted quinolizidine imides 21. Dihydro-2-pyridone compound 15, obtained previously from the aza-Diels-Alder reaction, was first oxidized to the sulfone 16 which was effectively converted to the azide 17. Further click chemistry of compound 17 with terminal alkynes provided regiospecifically the 1,4-disubstituted triazoles 18a-c. Sequential detosylation with Bu3SnH/AIBN, N-allylation and ring-closing metathesis (RCM) reaction then provided the bicyclic imides 21a-b.

Cocaine and amphetamine-regulated transcript (CART) peptides are neurotransmitters with important roles in drug abuse. The increase of CART expression in the brain induced by psychostimulants is associated with changes of behavior in addicted animals. We expressed and purified the single-chain variable fragments antibody (scFv) against CART55-102, and observed the effect of CART scFv on the expression of cocaine-induced behavior sensitization in mice. Results showed that the titer of CART scFv was 1.6 μg/ml. Single administration of CART scFv (intraperitoneal 0.04, 0.2, and 1 mg/kg) reduced the increasing locomotor activity induced by chronic cocaine intake in mice (P<0.05-0.01), but failed to affect the locomotor activity of naive mice. These results suggested that CART scFv may be a potential therapeutic tool to treat drug abuse.

Objective To investigate the effects of Ganmaidazao Decoction (GMDZD), Sinisan(SNS) and Sijunzi Decoction (SJZD) recipe on endogenous nerve growth factor (NGF) of rat with chronic stress.Methods In order to establish the rat models with chronic stress, the feet of animals were stimulated with a pat-tern of electrical impulses (long term, high voltage and constant current). The alterations of behavior of animals were observed and quantified by open-field and tail-suspension test. Levels of NGF in pitttitary,hypetholamus and blood plasma were quantified using radio-immunoassay (RIA) method. Results 1) In comparision with the wild-type (WT) control rats, locomotor activity decreased significantly in the models and groups treated by Chinese medicinal herbs (P<0.01). In addition,the Chinese medicinal herbs treated groups exhibited increased locomotor activity than the model groups (P<0.01). 2) The cross time of the model groups decreased significantly compared with the WT controls (P < 0. 01). Meanwhile, three Chinese medicinal herbs treated groups displayed longer cross time than the model groups (P< 0. 01). 3)The vertical movement times of the model groups decreased significantly compared with the WT controls (P < 0.01). Meanwhile, three Chinese medicinal herbs treated groups displayed in-creased vertical movement times than the model groups (P <0.05). 4)In the tail-suspension test, the duration of immobility increased significantly in the model groups compared with the WT controls (P < 0. 01). However, the duration of immobility of three Chinese medicinal herbs treated groups were less than the model groups (P < 0.01). 5) Compared to the model groups,the level of NGF in pituitary of animals treated by GMDZD and SNS in-creased significantly (P<0.01). In the hypothalamus,the level of NGF in SJZD treated groups decreased in com-parision with the WT control and model groups (P<0.05). Whereas, GMDZD and SNS treated groups decreased significantly than WT control and model groups (P<0.01). In the blood plasma,the level of NGF showed a signif-icant reduction in the model groups compared with the WT control. Conclusion The results suggest that the GM-DZD,SNS and SJZD recipe have positive effects on rat with chronic stress through regulating the level of endogenous NGF.

Objective To construct rat amniotic epithelial cells (AECs) modified with human basic fibroblast growth factor (bFGF) gene that combined with nerve growth factor (NGF) secreting peptide utilizing lentivirus vector, by which bFGF could be stably synthesized and secreted. Methods Amniotic epithelial cells were collected from the term rats by trypsin-treating process, and subcultured AECs were identified by immunocytochemistry and RT-PCR methods. Lentiviral vector with human bFGF gene was constructed, and lentivirus packaging was performed after DNA sequencing. Afterwards, AECs were infected with viral supernatant, then selected with blasticidin. Furthermore, the selected cells were detected for bFGF expression and secretion by immunocytochemistry, RT-PCR and ELISA methods. Thereafter, growth activity of AECs chimaera was investigated, and the biological functions of secreted bFGF was evaluated by culturing PC12 cells with conditioned medium of AECs chimaera. Results Immunocytochemical staining and RT-PCR showed that the rat AECs expressed epithelial-specific markers CK-19, neural cell markers nestin and GFAP, as well as pluripotent cells markers SSEA-4, Oct-4, Nanog, Sox2 and vimentin. The AECs chimaera sustainable expressing bFGF were obtained after infection and screening processes. Immunocytochemistry staining and RT-PCR showed that most of the AECs chimaera expressed bFGF, compared with the non-expression before transfection. ELISA results implicated that AECs chimaera were able to secrete bFGF peptide. AECs chimaera survived in the screening procedure grew much faster compared with common AECs. The conditional culture medium of AECs chimaera could stimulate PC12 cells growth and neurites development, as compared with AECs group. Conclusion bFGF gene could be transferred into rat AECs successfully by lentiviral vector, and the AECs chimaera could express and secret bFGF afterwards, indicating robust growth activity and significant neurotrophic activity.

目的:表达纯化可卡因-苯丙胺调节转录肽(CART)及其突变体,观察其对大鼠进食行为的影响.方法:PCR扩增CART活性片段CART55-102 cDNA,构建表达CART55-102原核表达载体,并对其68位和94位半胱氨酸残基突变,构建表达CART55-102(Cys68Ser)和CART55-102(Cys94Ser)原核表达菌株,经IPTG诱导表达后,亲和层析纯化3种小肽;行为学技术检测原核表达小肽对大鼠进食行为的影响.结果:获得的重组CART55-102及其突变体的纯度在95%以上,产物得率约75%;CART55-102可显著抑制饥饿大鼠的进食行为,而68和94位突变体对饥饿大鼠的进食行为无明显影响.结论:原核表达系统可以成功制备高纯度CART活性小肽和突变体,68位和94位突变导致CART抑制进食的作用消失.

Objective:To study the effect of cocaine-and amphetamine-regulated transcript(CART)protein vaccine on morphine analgesia and tolerance.Methods:The expression plasmid pGEX-4T3-CART was constructed by gene cloning.The CART protein was purified by glutathione s-transferase(GST)-affinity chromatography.The experiment included 6 groups: blank control,normal saline(NS),GST+Freund's adjuvant,and CART protein vaccine(5μg,10μg and 20μg)groups.After immunization for twice,all groups were tested in hot plate.Morphine analgesia effect was evaluated through s.c.injection with 6 mg/kg morphine solution,calculated by MPE%.Then morphine tolerance model was established,and the tolerance to morphine was tested by s.c.injection with 6 mg/kg morphine 12 h after the last injection.Results:CART vaccine itself had no pronounced effect on the pain threshold(P0.05).CART vaccine at 10μg significantly depressed the analgesic effect of morphine analgesia (P0.05).Compared with NS group,vaccine groups showed a potential antagonizing tolerance effect,especially in the 10μg group,with the MPE% significantly increased(P0.05).Conclusion..CART vaccine itself has no influence on the pain re- sponse;however,it can impair the analgesia effect of morphine and can antagonize the analgesia tolerance to morphine.

Corticotropin-releasing hormone (CRH) has been shown to exhibit various functions in hippocampus. In the present study, we examined the effect of CRH on the expression of serum/glucocorticoid-inducible protein kinase-1 (SGK-1), a novel protein kinase, in primary cultured hippocampal neurons. A dose-dependent increase in mRNA and protein levels of SGK-1 as well as frequency of SGK-1-positive neurons occurred upon exposure to CRH (1 pmol/l to 10 nmol/l). These effects can be reversed by the specific CRH-R1 antagonist antalarmin but not by the CRH-R2 antagonist astressin 2B. Blocking adenylate cyclase (AC) activity with SQ22536 and PKA with H89 completely prevented CRH-induced mRNA and protein expression of SGK-1. Blockage of PLC or PKC did not block CRH-induced SGK-1 expression. Our results suggest that CRH act on CRH-R1 to stimulate SGK-1 mRNA and protein expression in cultured hippocampal neurons via a mechanism that is involved in AC/PKA signaling pathways.

Aim:G protein-coupled inwardly rectifying potassium channels（GIRK）are im-portant for neuronal signaling and membrane excitability.In the present study,we intend to find whether GIRK channels express functionally in adult rat dorsalroot ganglion（DRG）neurons.Methods:We used RT-PCR to detect mRNA for4 subunits of GIRK in the adult DRG.The whole-cell patch clamp recordingwas used to confirm GIRK channels functionally expressed.Results:The mRNAfor the 4 subunits of GIRK were detected in the adult DRG.GTPγS enhancedinwardly rectifying potassium（K + ）currents of the DRG neurons,while Ba~2+inhibited such currents.Furthermore,the GIRK channels were shown to becoupled to the GABAB receptor,a member of the G protein-coupled receptorfamily,as baclofen increased the inwardly rectifying K + currents.Conclusion:GIRK channels are expressed and functionally coupled with GABA B receptorsin adult rat DRG neurons.

In our previous study, we proposed that there may be an unknown pathway in the upper stream of the known signal transduction pathway of Ciliary neurotrophic factor (CNTF) that mediates the neuro-protective function of CNTF In the present experiment, we observed that the neuro-protective function of the non-classic signal transduction pathway in a L-NMDA (a glutamic acid ion type receptor atagonist) induced hippocampal neuron injury model, using primary culture rat hippocampal neurons, continuous photography and gp130 immunohistochemical assay. The results showed that L-NMDA induced injurious reaction of hippocampal neurons, and CNTF was able to inhibit the toxic action of L-NMDA on hippocampal neurons. Additionally, when JAK/STATs in the known classic signal transduction pathway of CNTF were blocked by PTPi-2, the protective effect of CNTF against L-NMDA injury still existed. L-NMDA caused a rapid increase in the concentration of hippocampal intracellular free [Ca2+](i). CNTF was able to attenuate L-NMDA-induced elevation of [Ca2+](i), and blocking JAK/STATs in the known classic signal transduction pathway of CNTF did not affect L-NMDA-induced elevation of [Ca2+](i), indicating that, apart from the known classic signal transduction pathway, there may be some other transduction pathways for CNTF to exert the protective effect on hippocampal neurons, and this pathway is related to[Ca2+].

RET receptor signalling is essential for glial-cell-line-derived neurotrophic factor (GDNF)-induced survival and differentiation of various neurons such as mesencephalic neurons. To identify proteins that mediate RET-dependent signaling, yeast two-hybrid screening was performed with the intracellular domain of RET as bait. We identified a new interaction between RET and the adapter protein SH2-Bβ. Upon GDNF stimulation of PC12-GFRα1-RET cells (that stably overexpress GDNF receptor α1 and RET), wild-type SH2-Bβ co-immunoprecipitated with RET, whereas the dominant-negative SH2-Bβ mutant R555E did not. RET interacted with endogenous SH2-Bβ both in PC12-GFRα1-RET cells and in rat tissues. Mutagenesis analysis revealed that Tyr981 within the intracellular domain of RET was crucial for the interaction with SH2-Bβ. Morphological evidence showed that SH2-Bβ and RET colocalized in mesencephalic neurons. Furthermore, functional analysis indicated that overexpression of SH2-Bβ facilitated GDNF-induced neurite outgrowth in both PC12-GFRα1-RET cells and cultured mesencephalic neurons, whereas the mutant R555E inhibited the effect. Moreover, inhibition of SH2-Bβ expression by RNA interference caused a significant decrease of GDNF-induced neuronal differentiation in PC12-GFRα1-RET cells. Taken together, our results suggest that SH2-Bβ is a new signaling molecule involved in GDNF-induced neurite outgrowth.

先前我们曾提出,在已知的CNTF信号传导通路上游可能存在新途径介导其神经保护作用.运用原代培养大鼠海马神经元、活细胞连续照相、活细胞计数、活细胞内游离[Ca2+]测定及gp130免疫组化等方法,以Glu离子型受体激动剂L-NMDA诱发海马神经元损伤为细胞损伤模型,用JAK/STATs阻断剂PTPi-2阻断CNTF已知的经典信号传导途径,观察CNTF非经典信号传导途径的神经保护功能,并探讨其可能机制.结果表明,L-NMDA可引起海马神经元的损伤反应,CNTF可有效抑制L-NMDA对神经元的毒性作用; 使用PTPi-2阻断已知的CNTF经典信号传导途径中JAK/STATs后,CNTF保护海马神经元抵抗L-NMDA的损伤作用仍然存在.L-NMDA可引起海马神经细胞内游离Ca2+浓度([Ca2+]i)迅速升高,CNTF可显著减弱L-NMDA引起的[Ca2+]i升高,阻断CNTF的经典信号传导途径中JAK/STATs并不影响L-NMDA引起的[Ca2+]i升高,表明除了已知的经典信号传导通路,CNTF发挥保护海马神经元作用还有其他信号传导途径,这一途径与胞内[Ca2+]有关.

Objective To obtain recombinant neural cell adhension molecule L1(L1) and to study its structure and biological activity.Methods The cDNA encoding the mature rat L1 was isolated using RT-PCR from total RNA extracted from newborn SD-rat hippocampus tissue.The expression plasmid pETL1 mutants of several extracellular domains of(L1) were constructed by inserting L1 and its mutants cDNA into plasmid pET-28a(+) containing T7 promoter and transformed into E.coli BL21(DE3). A series expression strain BLL1 mutants were selected.Recombinant L1 and its mutant proteins were expressed at levels about 18.5%～30% of total bacteria protein in form of inclusion body after the induction.By Ni~(2+) chelation affinity chromatography,up to 90% L1's mutant proteins were purified.The expressed plasmid pcDNA3-L1 were transfected into PC12 cells and constructed PC12-engineered cells which stably and highly expressed the L1.Results Purified and refolded IgI-FN5、Ig(Ⅰ-Ⅵ) and FN(1-5) fragments could significantly promote the neurite outgrowth of PC12-engineered cells;fragment Ig(Ⅴ-Ⅵ) also can promote the neurite outgrowth but not soobvious as the before;fragment FN(3-5) have no function to induce the neurite outgrowth of PC12-engineered cells.Conclusions These results suggested that there are at least two segments in extracellular domains of L1 Ig(Ⅰ-Ⅵ) and FN(1-5) fragments are critical for inducing the neurite outgrowth of PC12-L1 cells and the key amino acids for signaling transduction located in the segments of Ig(Ⅰ-Ⅵ) and FN(1-5);fragment Ig(Ⅴ-Ⅵ) is necessary but not crucial for promoting the neurite outgrowth;fragment FN(3-5) is not important for L1 biological function.

Aim: To study the specific binding of PTB domain in Doks family on upstream molecular.Methods: EGFR was obtained by lysating COS 7 cell line that in vitro expressing EGFR. GST1PTB,GST4PTB,and GST5PTB were expressed by E. coli. Then PTB domain in Dok1,4, and 5 were identified whether bound to EGFR by GST pull down. Results: The PTB domain from Dok1 but not Dok4 or Dok5 could selectively bind to stimulated EGFR. Conclusion: There are distinctions between different Doks PTB domain and upstream molecular.

学科建设以人为本.搞好青年教师的培养是一项战略性任务,是实现学科可持续发展的保证.我室自1988年建立以来,重视师资队伍建设,尤其抓紧对青年教师的培养,现已形成了一支由中科院院士陈宜张教授领衔的老中青三结合、以青年为主、充满活力的师资队伍.青年教师已成为教学与科研工作的骨干力量,从而保证了教研室各项任务的顺利完成.

目的观察睫状神经营养因子(CNTF)及其受体CNTFRα在大鼠眼球、泪腺上的分布情况.方法取雄性SD大鼠两侧眼球和泪腺,作石蜡切片,用免疫组织化学ABC法染色检测眼球、泪腺中CNTF和CNTFRα的免疫阳性反应.结果 CNTF和CNTFRα的免疫阳性反应产物在眼球和泪腺的组织定位基本相同,包括角膜上皮细胞、固有层神经纤维、角膜细胞、内皮细胞,虹膜上皮细胞,睫状体上皮细胞,晶状体上皮,视网膜色素上皮细胞、苗勒细胞、节细胞层细胞,球结膜上皮细胞,泪腺腺细胞及导管上皮.结论 CNTF及其受体选择性地分布于眼球和泪腺的一些细胞中,且多数细胞与房水和泪液的产生和接触有关.

Objective:To observe the effect of ciliary neurotrophic factor (CNTF) on the increase of hippocampal neurons -[Ca~(2+)-]_i induced by thapsigargin (Tg), a sarco/endoplasmic reticulum Ca~(2+)-ATPase (SERCA2a) inhibitor. Methods: The primarily cultured hippocampal neurons of newborn SD rats(within 24 h of delivery) were prepared after 10-14 d culture. The intracellular -[Ca~(2+)-]_i was measured with Fura-2/AM in real time. The neurons were divided into 3 groups. Group A (n=20), EGTA (3 mmol/L);Group B (n=40), EGTA (3 mmol/L) + Tg (10 μmol/L);and Group C (n=12), EGTA (3 mmol/L) +Tg (10 μmol/L) +CNTF (500 U/μl). Results: When the extracellular Ca~(2+) was chelated by enough EGTA, -[Ca~(2+)-]_i was ≤(30.73±7.25) nmol/L; after EGTA+Tg was added, -[Ca~(2+)-]_i significantly increased(-[Ca~(2+)-]_i≥-[91.55±12.24-] nmol/L); and after EGTA+Tg+CNTF was added, -[Ca~(2+)-]_i was markedly reduced compared with that in group B (-[Ca~(2+)-]_i≤-[26.09±7.16-] nmol/L, P0.01). Conclusion: CNTF can significantly decrease the inhibitive effect of Tg on SERCA2a and thus reduce the cytoplasm -[Ca~(2+)-]_i, suggesting that CNTF may improve the uptaking of -[Ca~(2+)-]_i from cytoplasm and reduce the Ca~(2+) load in hippocampal neurons.

神经营养素(neurotrophin, NT)受体具有低亲和力的Mr为75 000 NT受体(75 KD NT receptor, p75NTR)和高亲和力的原肌球蛋白受体激酶(tropomyosin receptor kinase, Trk)受体家族两类.二者均为跨膜蛋白受体, 参与调节以神经元为主的某些细胞的生长、分化、存活、修复以及凋亡等多种生物学效应.研究表明, 二者间的信号转导及生物学效应既相互协同又彼此拮抗, 既紧密联系又有显著区别.Trk受体一般介导"正性"信号, 如促进神经元生长、维持其存活; 而p75NTR则具有多种生物学效应, 除可促进神经元存活、生长外, 还可诱导神经元凋亡、抑制神经元轴突生长以及参与细胞周期的调节, 即可介导"正性"和"负性"两种效应, 但以介导"负性"促凋亡效应为主, 这与p75NTR所在细胞的类型、生理与功能状态、发育阶段以及局部环境有关.