Objective To screen a key molecule, leucine-rich glioma inactivated protein 4 (LGI4), which participates in endothelial cell-mediated osteosarcoma metastasis, and to further analyze its function and molecular mechanism in osteosarcoma metastasis. Methods A co-culture model of human umbilical vein endothelial cells (HUVECs)-osteosarcoma cells was established, and the genes with significant fold change were screened by whole transcriptome sequencing in combination with relevant literature. After, the candidate target gene, LGI4 was verified with real-time quantitative PCR (RT-qPCR) and Western blotting, the expression of LGI4 at mRNA and protein levels was also determined. Osteosarcoma cell line 143B and MTF LGI4 were transfected with the overexpression plasmid of LGI4 to construct the cells with stable overexpression of LGI4. Then the experiments included 5 groups of osteosarcoma cells, including negative control group (normally cultured), co-culture group (co-cultured with HUVECs), co-culture medium group (treated with the HUVECs medium), vector control group (transfected with control plasmid) and LGI4 group (overexpression of LGI4). Transwell assay and wound healing test were used to detect the migration and invasion abilities of osteosarcoma cells. A tumor metastasis model in nude mice was used to verify the effect of LGI4 overexpressed osteosarcoma cells on distant metastasis in vivo. Bioinformatics analysis was applied to analyze the potential downstream target signaling pathways of LGI4, and recovery experiments were performed with related inhibitors to clarify the role of target signaling pathways in the regulation of osteosarcoma metastasis by LGI4. Results The expression levels of LGI4 were markedly decreased in 143B and MTF cells after co-culture with HUVECs (P < 0.05). Compared with the negative control group, the cell migration and invasion abilities of the co-culture group and co-medium group were significantly enhanced, whereas the cell migration and invasion rates of the LGI4 overexpression group were significantly lower than that of the vector control group (P < 0.05). The nude mice metastasis model revealed that the metastasis ability of osteosarcoma cells was suppressed with LGI4 overexpression. Further analysis revealed that NOTCH4 signaling pathway was the downstream target signaling pathway of LGI4, and functional recovery experiments confirmed that LGI4 resulted in osteosarcoma metastasis by inhibiting NOTCH4 signaling pathway (P < 0.05). Conclusion Endothelial cells activate NOTCH4 signaling pathway and thus promote tumor metastasis by inhibiting the expression of LGI4 in osteosarcoma cells.

Objective To investigate the difference of inner ear ultrastructure in acute otitis media induced by different virulent bacteria. Methods Sixteen guinea pigs were randomly divided into two groups. The right middle ears were inoculated with 0.1ml 1×108/ml staphyloccoccus aureus or pseudomonas aeruginosa respectively. The left ear serves as control. On the 3rd day after infection, alteration of the cochlea was observed by scaning and transmisson electron microscopy. Results On the 3rd day after infection, the damage of cells and degeneration of organelles could be observed in both groups of staphyloccoccus aureus and pseudomonas aeruginosa. The alterations of ultrastructure in the pseudomonas aeruginosa-infected group are more apparent than those in the staphyloccoccus aureus-infected group. Conclusion There are morphological changes of the inner ear in early acute otitis media. The acute otitis media infected by pseudomonas aeruginosa may induce serious sensorineural hearing loss.

Aim: To study the changes of T-lymphocyte subsets in regenerated tissue from autosplenic graft of patients with idiopathic thrombocytopenic purpura(ITP) and its effects on immue function. Methods: Autosplenic tissue was transplanted into the omental sac in 12 cases of ITP.In the 6th month after transplantation,biopsy specimen of the regenerated splenic tissue was collected. T-lymphocyte subsets of the splenic tissue was analyzed by immunohistochemical APAAP technique and image analysis. Results: In regenerated splenic tissue,CD 3 positive cells remarkably decreased in marginal zone and periarterial lymphatic sheath (PALS)( P0.05),and significantly increased in RP(P0.01).CD 4 positive cells significantly decreased in germinal centers and PALS(P0.01).CD 8 positive cells remarkably increased in PALS and RP(P0.01). Conclusion: The distribution of T-lymphocyte changes in regenerated spleen and is benefit to inhibit the PAIgG.

目的测定肌肉注射盐酸川芎嗪注射液后川芎嗪在豚鼠血液、脑脊液和耳蜗外淋巴液中的分布.方法豚鼠经肌肉注射盐酸川芎嗪注射液(2 ml/kg)后,采用高效液相色谱法(HPLC)中的内标法和外标法测定血液、脑脊液和耳蜗外淋巴液中川芎嗪的浓度.结果川芎嗪注射后,豚鼠血液中川芎嗪浓度迅速升高,20 min达到最大值(357.76 μg/ml),2 h后血药浓度迅速下降;在脑脊液中10 min后才检测到川芎嗪,20 min其浓度达到最大值(120.50 μg/ml),70 min后脑脊液药浓度迅速下降;在耳蜗外淋巴液中5 min时能检测到川芎嗪, 50 min时浓度达最大值(215.79 μg/ml),70 min后药物浓度明显降低.结论川芎嗪可吸收入血液并能够通过血脑屏障及血-迷路屏障进入脑脊液和耳蜗外淋巴液,药物吸收及消除迅速.临床应用该药时,要维持川芎嗪在耳蜗内的药物浓度应该适当增加用药次数,以提高疗效.

目的观察层粘连蛋白(Laminin,LN)和纤维连接蛋白(Fibronectin,FN)在乳腺不典型增生及乳腺癌中表达的变化.方法用S-P免疫组化染色法分别检测正常乳腺组织、Ⅰ、Ⅱ、Ⅲ级不典型增生的乳腺组织及乳腺癌组织中LN和FN的表达情况.结果正常乳腺组织中LN只在基底膜表达;FN表达于基底膜及少量间质细胞.Ⅰ级增生的乳腺组织中LN主要表达于基底膜及上皮细胞;FN表达近似正常组织.Ⅱ级不典型增生中LN于上皮细胞表达增多;FN主要表达于基底膜及上皮细胞.Ⅲ级不典型增生中LN的间质表达明显增多(P<0.05),而上皮细胞表达明显减少(P<0.05);FN则主要表达于上皮细胞及增生腺管周围的基质中.浸润性导管癌中LN主要表达于癌细胞,间质表达显著减少(P<0.05);FN亦主要表达于癌细胞中.结论乳腺不典型增生时LN表达部位的变化反映了乳腺上皮细胞的增生状态,不典型增生的乳腺组织中间质FN的表达是机体的一种防御反应.

目的总结脾部分切除术治疗脾脏外伤的临床经验.方法对本院16年间105例实施脾部分切除治疗脾外伤的病人进行回顾性分析,重点分析脾部分切除术的适应证、手术方法和临床疗效.结果 67例(63.8%)为单纯性脾脏外伤.按脾脏损伤程度分级标准,Ⅰ级脾外伤13例(11.4%),Ⅱ级56例(53.3%),Ⅲ级28例(26.7%),Ⅳ级8例(7.6%).术式包括:脾段切除4l例、半脾切除35例、次全脾切除29例,对儿童脾外伤附加自体脾组织移植21例.全组病例无手术死亡,术后膈下积液和感染3例,均经保守治疗治愈.结论对部分脾脏外伤病人的治疗选择脾部分切除术是安全可行的.

Objective To explore the expressions of tissue inhibitor of metalloproteinase (TIMP) and matrix metalloproteinase (MMP) in the spleen of cirrhotic rats with splenomegaly and hypersplenism before and after subtotal splenectomy. Methods Cirrhosis of liver was induced with 60% carbon tetrachloride and 5% alcohol in rats, then subtotal splenectomy was performed. The levels and cellular locations of TIMP1, MT1 MMP, MMP9, and MMP2 in the spleen of splenomegaly and hypersplenism before and after subtotal splenectomy were determind with immunohistochemistry and in situ hybridization. Results The amounts of TIMP1, MT1 MMP, MMP9, and MMP2 expressions had no significant differences in the spleen of cirrhotic rats with splenomegaly and hypersplenism before and after subtotal splenectomy ( P 0.05). Conclusion Subtotal splenectomy did not enhance the fibrosis of the residual splenic tissue.

目的观察大鼠甲状腺髓样癌细胞系CA-77在不同培养条件下生长、增殖、降钙素(CT)及降钙素基因相关肽(CGRP)合成表达等情况,探讨CA-77细胞的培养条件. 方法分别采用DMEM,RPMI 1640和DMEM/Ham's F12(1∶1)含血清培养液及无血清培养液进行CA-77细胞培养,分别绘制细胞生长曲线和CT及CGRP的免疫组化染色. 结果 CA-77细胞在DMEM,RPMI 1640和DMEM/Ham's F12(1∶1)含血清培养液和无血清培养液中均可保持其良好的生物特性. 结论 CA-77可以在常用培养液中培养,并可在无血清条件下保持稳定的生物特性.

Objective To investigate the effects of subtotal splenectomy on liver fibrosis. Methods After liver fibrosis was induced with 60% carbon tetrachloride and 5% alcohol in rats, the rats were randomized into 4 groups of operative liver fibrosis (OLF), total splenectomy (TS), subtotal splenectomy (SS) and sham operation (SO). The values of serum PCIII, HA, LN and PLD were determined. Meanwhile, the level and cellular sites of gene expression of TGF-β1, MT1-MMP, MMP2 and TIMP1 were detected with immunohistochemistry and in situ hybridization. Results No significant differences in levels of PCIII, HA, LN and PLD were found between the TS group and SS group (P0.05). However, the levels were markedly higher in rats with liver fibrosis than in the controls (P0.0001). The expression of TGF-β1, MT1-MMP, MMP2 and TIMP2 occurred predominantly in activated myofibroblasts, fat-storing cells. Image analysis showed that the level of TGF-β1 in fibrotic cells was lower after than before the operation (P0.0001). However, it was still significantly higher than that in the controls (P0.0001). After operation, the level was not remarkably different among the 4 experimental groups (P0.05). The level of expression of MT1-MMP, MMP2 and TIMP1 was significantly higher in the experimental groups than in the control (P0.0001). But there were no marked differences in their expression in 1 group before and after the operation and among the 4 groups after the operation (P0.05). Conclusion The residual splenic tissue after subtotal splenectomy does not aggravate the liver fibrosis.

Objective To investigate the role of platelet activating-factor (PAF) and its antagonist in serious intra-abdominal infection (SIAI) of rats, and the relationship between PAF and TNF or IL-8. Methods In a ligated and perforated cecum model of septic rat. The mortality and the level of blood PAF, serum TNF, IL-8 at different time points were observed. Results ①The level of blood PAF, serum TNF and IL-8 increased with a sig- nificant correlation between PAF and TNF/IL-8. ②After treated with PAF antagonist WEB2170, the level of blood PAF, serum TNF and IL-8 decreased with a reduced mortality rate. Conclusion The PAF antagonist WEB2170 can block the communication network of PAF with TNF or IL-8 and plays an important role in preventing MOF.

目的了解充血性脾肿大伴脾功能亢进(脾亢)患者血小板相关抗体(PA-IgG)水平及不同脾切除术后的改变,探索脾肿大、血小板、PA-IgG之间的关系.方法采用竞争性酶联免疫吸附试验(ELISA)检测了24例脾肿大伴脾亢患者血清PA-IgG水平.结果脾肿大伴脾亢患者的PA-IgG水平明显高于正常者(P＜0.01),而血小板值低,PA-IgG与血小板之间存在显著负相关(r＝-0.4747,P＜0.05).脾次全切除和脾切除术后,PA-IgG水平下降,血小板上升,两者之间的负相关关系消失.结论机体内可能存在PA-IgG与血小板的免疫调节机理,而脾脏可能与此有关.

1982年以来,我院共收治经手术和病理证实的脾占位性病变31例,现将其诊断和治疗体会报告如下.

目的 探讨脾大部切除术治疗肝硬化脾大脾亢的疗效。方法 对 2 6例肝硬化脾大脾亢病人行脾大部切除加断流术 ,术后 1年内随访 ,观测门静脉压力和直径、外周血细胞计数、骨髓细胞学、血小板相关IgG（PAIgG）及残脾大小的变化。结果 脾大部切除加断流术后 ,门静脉压力下降 ,门静脉直径缩小 ,残脾显像良好 ,无明显增大 ,外周血红细胞、白细胞和血小板计数升高 ,骨髓像大致正常 ,PAIgG下降 ,随访 1年见脾亢明显改善或消失。结论 脾大部切除术治疗肝硬化脾大脾亢疗效可靠 ,术后残脾无明显增大 ,保留略小于正常脾大小的残脾是适宜的。

目的:探讨原发性血小板减少性紫癜(ITP)患者自体脾移植后再生脾的免疫结构变化及其对原发病的影响.方法:对12例ITP患者行脾切除自体脾移植术,术后6月活检再生脾组织,应用ABC免疫组化和图像分析,结合透射电镜对脾组织的形态结构、巨噬细胞和树突状细胞等主要免疫细胞成份进行观察.结果:再生脾具有与正常脾相似的形态结构,但滤泡面积及数目、生发中心数目、巨噬细胞和树突状细胞均显著少于移植前脾及正常脾;超微结构见再生脾浆细胞数及巨噬细胞吞噬现象均较移植前减少.结论:ITP患者自体移植再生脾具有部分免疫吞噬功能;再生脾免疫结构发生了重建,不会因脾再生而致原发病复发.

对我院1992～1995年收治的175例低位直肠癌外科治疗行回顾性分析,就肿瘤术式选择及预防术后复发问题讨论如下.

目的:了解膜型基质金属蛋白酶1(MT1-MMP)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶组织抑制剂1(TIMP1)在大鼠正常及纤维化肝脏中的表达.方法:用60%CCl4加5%乙醇制作大鼠肝纤维化模型,利用原位杂交及免疫组化的方法检测MT1-MMP, MMP2,TIMP1的表达并与正常对照组比较.结果:在纤维化肝脏中,MT1-MMP, MMP2,TIMP1主要表达在成纤维细胞、肌成纤维细胞中,以纤维间隔及汇管区最明显,血管内皮细胞有低水平表达.而正常组中仅有内皮细胞有低表达,明显弱于肝纤维化组.结论:在肝纤维化中,成纤维细胞及肌成纤维细胞是MT1-MMP、 MMP2、TIMP1表达的主要细胞.

为观察Tenascin(TN)在乳腺不典型增生癌变过程中的表达变化,采用SP免疫组化染色法检测了50例不同乳腺组织中TN的表达情况.结果显示: 正常及Ⅰ级增生的乳腺组织中无TN表达,2例Ⅱ级不典型增生的乳腺组织中有TN表达,Ⅲ级不典型增生及浸润性导管癌中TN表达阳性率分别为80%和90%,与Ⅱ级不典型增生相比明显增高(P＜0.01).部分癌细胞胞浆中也有TN表达.本实验结果提示,重度不典型增生的乳腺组织间质中TN的表达可能起限制不典型增生细胞向外生长的作用.

Objective: To study the long-term therapeutic efficacy of splenectom y and splenic autotransplantation forpatientswith idiopathie throm bocytopenicpurpura(ITP). Methods: 24casesofITPw ere treated with splenectomy and splenic autotransplantation and follow ed up for 60 to 96 m onths. Results: Therew as a significantincrease ofplateletcountone week postoperatively and a m arked decrease ofIgM, IgG and lym phocyte transform ation rate in the first m onth after operation. The platelet count, IgM, IgG and lym phocyte transform ation rate remained norm alin the follow -up period. The size ofthe transplanted spleen was 5 cm ×6 cm in allthe cases. Gam mascintigraphy dem onstrated that the splenic tissuespossessed phagocytic function. Conclusion: Our findings suggestthatautotransplantation ofsplenic tissue notonly treats ITPbutalso preserves a partofthe splenic functions. In addition, itw illnotinduce therecurrence ofITP.

目的通过系列的动物实验和临床研究,达到在脾损伤时采用多种方法保留脾功能。方法在动物实验成功的基础上,应用保脾术于临床,近20年我们收治脾损伤263例,其中全脾切除83例（31.56%）;采用多种保脾技术180例（68.44%）。后者行切脾后自体脾移植90例,脾修补和部分切除45例,脾动脉单纯结扎或保留副脾10例,非手术疗法保脾35例;脾修补的方法是用4号丝线作褥式或间断缝合;脾移植是取脾的25%切成1cm×1cm×0.5cm 块状,植入网膜袋中。结果保脾组死亡6例,占手术保脾组的4.14%,死因与保脾技术无关。存活的保脾病例随访最长15年,B 超和核素锝扫描显示术后3个月脾修补裂伤愈合,脾移植的脾块显像良好;脾移植与全脾切除比较,血清Tuftsin 显著上升,痘痕红细胞计数明显下降。结论临床上采用的多种保脾技术,包括自体脾移植均能恢复脾的生理功能。