去细胞化肝脏生物支架材料的制备

目的探讨制备去细胞化肝脏生物支架(decellularize d liver biological scaffold,DLBS)的新方法,寻找适合肝脏组织工程的新型支架材料。方法 (1)采用化学去垢剂—酶联合去细胞化技术制备去细胞化全肝脏生物支架,并观察去细胞化效果;(2)DLBS与C3A及骨髓间充质干细胞体外共培养,观察DLBS细胞相容性。(3)通过四氮唑盐比色法(MTTAssay)观察DLBS对C3A的增殖作用。(4)将DLBS植入SD大鼠背部皮下,4周后处死大鼠,观察鉴定DLBS的组织相容性。结果通过HE染色、扫描电镜观察证实经过化学去垢剂—酶联合去细胞方法制备的去细胞化全肝生物支架不含细胞成分,只剩下胶原、弹性蛋白等支架成分;体外培养实验发现,L02及骨髓间充质细胞能够与DLBS粘连、生长。MTTAssay检测证实DLBS有促进C3A生长、增殖的作用。结论利用化学去垢剂—酶联合去细胞化技术制备的DLBS脱细胞彻底、细胞外基质保留较完整,并且有促进细胞粘附、增殖和分化的作用,是一种较为理想的生物支架材料。     

去细胞化技术在全肝生物支架建立中的应用

目的 通过去细胞化技术建立保留细胞外基质的完整肝脏支架,并对支架进行形态结构和保留成分鉴定.方法 通过门静脉插管,依次灌注去垢剂曲拉通X-100(Triton X-100),十二烷基硫酸钠(SDS),并用磷酸盐缓冲液(PBS)洗脱残留去垢剂,对所得支架进行HE染色、门静脉及月日道铸型、扫描电镜、纤维成分染色鉴定等形态学观察.并将支架切成50 μm的厚度后与C3A细胞系共培养,进行生物相容性验证.结果 经本实验方案得到肝脏生物支架的成功率为75%.肝脏生物支架包膜完整,肉眼可见肝脏内Gllisson管道结构.HE染色示无细胞及胞核残存.管道铸型见胆道、门静脉完整,无铸型液溢出.扫描电镜示大量纤维网状结构存在.纤维成分染色见大量胶原纤维、弹力纤维存在.生物相容性实验初步显示该支架具备较好的生物相容性,可以作为生物支架材料应用.结论 经本实验去细胞化过程,肝组织细胞可从细胞外基质中完全洗脱下来,并保留比较完整的肝脏管道系统.本研究获得全肝生物支架可以作为肝脏器官培养的基础支架.     

适配子修饰去细胞瓣支架促进再内皮化

摘要:目的 研究将适配子固定在去细胞瓣支架表面加速瓣膜再内皮化的作用。方法 制备3组主动脉瓣膜支架: 去细胞瓣组、适配子固定的去细胞瓣组(适配子组)、戊二醛交联的去细胞瓣组(戊二醛组)。用流式细胞仪检测适配子与 内皮祖细胞的结合效率;荧光显微镜检测适配子在去细胞瓣表面的固定;通过混合培养粘附实验检测内皮祖细胞在支架 上的粘附和增殖能力。结果 适配子与内皮祖细胞的结合实验显示适配子对内皮祖细胞具有特异性;荧光显微镜显示 适配子被固定于去细胞瓣支架上;细胞培养实验显示适配子组支架上粘附的内皮祖细胞数量高于其余两组支架并且增 殖较快。结论 适配子能够促进内皮祖细胞在去细胞瓣支架上的特异性粘附和增殖,从而促进再内皮化。     

小鼠胰岛与肝脏脱细胞支架三维共培养的实验研究

目的:观测原代小鼠胰岛在小鼠肝脏脱细胞支架上的生长及功能,探讨组织工程学治疗糖尿病的新方法?方法:运用灌注法制备结构完整的全肝脏脱细胞支架并检测,运用胶原酶P灌注消化法获取小鼠胰岛细胞并行结构和功能完整性分析;将分离纯化的原代小鼠胰岛经门静脉灌入全肝脏脱细胞支架,在三维培养体系培养后进行相关检测?结果:肝脏脱细胞支架检测未见细胞结构,胶原结构保存完整?支架内剩余DNA定量检测结果为(38 ± 11)ng/mg dsDNA,生物相容性良好?双硫腙(dithizone,DTZ)染色胰岛呈现特异性猩红色,葡萄糖刺激实验提示在高糖组中培养的原代小鼠胰岛胰岛素分泌功能明显高于低糖组,差异具有统计学意义(P < 0.01);将胰岛移植入肝脏脱细胞支架培养5 d后胰岛定植,且胰岛素基因表达显示脱细胞支架三维培养中胰岛素表达水平与平面培养相比,差异具有统计学意义(P < 0.01)?结论:肝脏脱细胞支架三维培养体系中的胰岛细胞比传统平面培养具有更佳的细胞活力及更有效的胰岛素分泌功能?     

肝癌细胞铁蛋白磁共振成像的实验研究

目的 研究肝癌细胞中内源性铁蛋白的表达情况,并进一步分析铁蛋白的表达与磁共振成像（MRI）的信号关系如何。方法 对3种肝癌细胞株QGY-7703、HepG2、SK-Hep-1以及正常肝细胞L02,4种细胞株内源性铁蛋白表达情况进行检测,用RT-PCR方法 检测基因表达,Western blot检测细胞株中铁蛋白表达,同时测定细胞培养液上清中铁蛋白的分泌量。最后检测4种细胞磁共振信号的情况,分析细胞铁蛋白表达与磁共振信号的关系。结果 RT-PCR与Western Blot均显示铁蛋白的基因表达与蛋白表达,HepG2与QGY-7703表达最强,SK-Hep-1表达中等,L02表达很弱。4种细胞培养液上清中HepG2与QGY-7703分泌铁蛋白量较高,SK-Hep-1分泌较低,L02分泌量很低。磁共振细胞显像,T1WI、T2WI及PD均显示HepG2与QGY-7703磁共振信号强度低,而SK-Hep-1与L02信号强度较高。结论 各种肝癌细胞中铁蛋白表达水平不同,铁蛋白表达水平与磁共振细胞显像信号强度呈负相关。     

大鼠子宫去细胞化支架的制备和评价

目的：通过去细胞化灌注技术,探讨成功制备天然子宫去细胞化生物支架的理想灌注策略,并通过系统的评价体系,以鉴定其能否作为子宫组织再生工程研究的良好应用载体。方法：选择健康成年雌性SD大鼠,采用子宫动脉入路途径,通过冻融、酶解及曲亚通（TritonX-100）联合十二烷基硫酸钠（SDS）的洗脱灌注等流程,获取子宫去细胞化支架。并通过大体形态观察、美兰染色、HE染色观察、基因组DNA定量分析、EGF、bFGF、TGF-β含量测定、电镜观察、胶原蛋白总量检测及主要胶原成分鉴定等对去细胞化后的子宫支架进行全面的评价。结果：成功建立动脉灌注入路并获得了肉眼透明的子宫去细胞化支架,HE染色和透射电镜观察均表明去细胞化子宫支架内无细胞残留,DNA含量检测表明支架内DNA残留量为（45.6±7.3）ng/mg,不足正常子宫的5%（P＜0.01）,美兰染色和扫描电镜显示子宫支架的脉管网络和空间结构保存完整;而胶原蛋白含量由新鲜子宫组织的（0.57±0.12）μg/mg增加到去细胞化子宫支架的（0.88±0.10）μg/mg,差异有统计学意义（P＜0.05）,结合各型胶原的免疫组织化学定性分析,说明去细胞化后子宫支架的细胞外基质成分保留得较为完整;ELISA结果显示去细胞化子宫支架中细胞因子EGF、bFGF和TGF-β分别保留了66%、85%和54%,表明其仍具备一定的生物活性。结论：本研究方法能够成功制备理想的去细胞化子宫支架,并且支架的基质成分和理化特性完整保留,能够作为子宫组织工程研究的良好载体。     

混合细胞共微囊化对肝细胞功能的支持作用

目的观察大鼠肝细胞、转基因肝星状细胞株HGF／CFSC和／或大鼠骨髓来源Thy-1^＋β2M^-细胞（BDTC）共微囊化对肝细胞生物学活性的支持,及腹腔移植混合细胞微囊对急性肝衰竭大鼠肝功能的改善作用。方法利用微囊发生器制备含肝细胞或混合细胞的微囊,依微囊内包裹细胞种类不同,分为微囊化肝细胞组、微囊化肝细胞＋CFSC／HGF组）和微囊化肝细胞＋CFSC／HGF＋BDTC组,通过观察囊内细胞形态和体外培养测定培养液中白蛋白和尿素的分泌,判断各组囊内肝细胞活性和功能的维持;将90％肝大部切除所致的急性肝衰竭大鼠按照移植微囊种类不同分为空囊对照组和上述3个实验组（每组10只）,观察腹腔植入后不同时间大鼠的一般状况、存活时间、血生化改变、肝组织再生及微囊化移植物的组织学特征。结果与单独肝细胞微囊者相比,混合细胞微囊内肝细胞存活时间超过1倍,培养液中白蛋白分泌和尿素合成量明显增加（均P〈0．01）;与对照组相比,微囊化肝细胞或微囊化混合肝细胞移植后,急性肝衰竭大鼠的肝功能显著改善、存活率明显提高（10／10 vs 1／10）,其肝组织再生完全;移植21—42d时,部分微囊附着于肝脏表面并出现血管化,微囊表面存在不同程度的纤维化,微囊内仍有存活的细胞,以微囊化混合肝细胞组优于微囊化肝细胞组。结论混合细胞共微囊化能明显改善囊内肝细胞的存活寿命、形态和功能的维持,微囊化混合肝细胞腹腔移植对促进急性肝衰竭大鼠的肝功能恢复具有显著作用。     

人肝组织脱细胞支架的制备

目的探究人肝组织脱细胞支架的制备。方法本研究利用手术切除的人肝血管瘤左外叶组织,经反复冻融,0.01% SDS、
0.1% SDS和1% Triton X-100循环灌注制备人肝组织脱细胞支架,并通过灌注过氧乙酸消毒。将L-02细胞通过门静脉插管种
植于脱细胞支架内进行培养。结果HE、DAPI染色和扫描电镜结果显示脱细胞支架内无细胞成分残留,残余DNA检测为
25.3±14.6 ng/mg干质量,小于新鲜肝脏DNA含量的1%。免疫组化证实支架内保留了Ⅰ型胶原、Ⅳ型胶原、纤连蛋白、弹力蛋白
成分。L-02细胞在支架上生长良好且有增殖,并表达白蛋白及葡萄糖-6-磷酸酶。结论利用手术切除的人肝标本行肝组织脱
细胞支架的制备是可行的,且为构建更适用于临床的组织工程肝脏提供了新思路。
     

瘦素及其受体蛋白在肝癌细胞中的表达及意义

目的:研究瘦素及其受体在人肝细胞癌细胞株中的表达情况,探讨其在肝癌发生过程中的可能作用。方法:制备人正常肝细胞株L02及人肝细胞癌细胞株HepG2、SMMC7721的细胞爬片,采用免疫组织化学染色法检测上述细胞株中瘦素和瘦素受体蛋白的表达,以医学图文分析系统进行定量分析。结果:人正常肝细胞株L02及人肝细胞癌细胞株HepG2、SMMC7721中均存在瘦素及其受体蛋白的阳性表达,并且瘦素及其受体蛋白的表达水平均是人肝癌细胞株高于人正常肝细胞株,差异具有统计学意义(P<0.01)。结论:瘦素及其受体以双重表达方式存在于人肝细胞癌细胞中,可能在肝癌的发生中起到一定作用。     

骨膜去细胞生物支架在骨缺损小鼠体内的血管化机制

目的：观察骨膜去细胞生物支架对小鼠股骨骨缺损的修复作用及支架内血管形成过程,探讨其血管形成的可能机制。方法：采用物理冻融、化学和生物酶试剂等序贯处理获得骨膜去细胞生物支架。体外实验方面,通过细胞划痕试验观察骨膜去细胞支架浸提液对人脐静脉内皮细胞（HUVEC）迁移的影响以评价骨膜去细胞支架中是否存在促血管化的生物因子,同时通过酶联免疫吸附试验（ELISA）检测支架中的血管内皮生长因子（VEGF）。在体动物实验方面,通过建立小鼠股骨骨缺损模型评价骨膜去细胞生物支架通过促血管化诱导骨修复的可能性。在小鼠股骨远端制备0.5 mm直径的单皮质骨缺损后,于骨缺损处植入骨膜去细胞支架后逐层缝合切口,对照组小鼠骨缺损处不放置材料。分别在术后第7天、第14天、第21天和第28天,取材、固定、脱钙、包埋和切片,通过HE染色评价骨缺损区骨修复情况,通过免疫荧光染色观察血管性血友病因子（vWF）以评价缺损区血管化情况。结果：体外细胞实验表明,去细胞骨膜支架浸提液对HUVEC的增殖没有明显的抑制作用;细胞划痕实验结果显示,与对照组相比,支架浸提液组细胞的迁移面积更大,去细胞骨膜支架浸提液能够有效促进HUVEC的迁移并且在支架浸提液中检测到VEGF,其浓度为210 pg/mL。动物实验方面,HE染色证明去细胞骨膜支架可以促进骨缺损区域血管的生长和新骨形成,免疫荧光染色进一步证明去细胞骨膜支架对骨缺损的修复活动伴随血管化的发生过程,且去细胞骨膜支架中血管的密度随时间延长呈现先增多后减小的趋势。结论：去细胞骨膜支架可以血管化,并且促进骨缺损的愈合。VEGF可能是其血管形成过程中的关键因素。     

APPLICATION OF LORNOXICAM TO PATIENT-CONTROLLED ANALGESIA IN PATIENTS UNDERGOING ABDOMINAL SURGERIES

FOR anesthesiologis s ,treatingpostoperativepainhas alwaysbeen a problem.Althoughopioidshave been provedtobe effective,theirsideeffectscouldnotbeignored.With thedevelopmentofscienceand pharmacology,many drugs with aspectsof satisfactoryanalgesicefficacyand couldbe welltoleratedby patientshave been developed.And lornoxicamisone of them, which isa non-steroidalanti-inflammatorydrug (NSAID ), with analgesic, anti-infl-ammatory,andantipyreticproperties.Itseliminationhalf-time(3 to 5 hours) isle…     

Acupuncture Treatment of Vomiting

CASE HISTORY A female patient, 46 years old, head of the foreign affairs department of a certain university in Beijing, paid her first visit on October 9, 2006, with the chief complaint of vomiting for one month. She got vomiting after meals in early September. Before that, she had discomfortable sensation in the stomach due to angry with others, but she didn't pay much attention. Later, it developed into vomiting after eating. After the vomiting, the discomfort would be relieved, but with slight hypodynamia. She was once diagnosed as having 'neurogenic vomiting'. Having taken some western and Chinese drugs, the above symptoms were a little bit improved, but she would have nausea upon eating and with regurgitation. Because of the fear for vomiting, she did not dare to have food intake, with body weight reduction of 6 kilos in one month.     

Qiu Baoguo''''s Experience in Treating Consumptive Syndromes after Radio-Chemotherapies

Radiotherapy and chemotherapy are the important modern medical therapies for malignant tumors,yet they can also bring about serious local and systemic toxic side reactions so to decrease the patient;'s life quality,manifested by a series of consumptive symptoms.Having engaged in the combined work of Chinese and western medicine for nearly 50 years,the research fellow Qiu Baoguo in Henan Provincial Academy of TCM has developed his unique views on the TCM study of consumptive syndromes.The author of this essay had once the fortune tO follow Dr.Qiu in clinic,and specially would like to introduce in the following Dr.Qiu's experience in treating consumptive syndromes after radio-chemotherapies for patients with malignant tumor.     

Insomnia Due to Deficiency of Both the Heart and Spleen Treated by Acupuncture-Moxibustion and Chinese Tuina

OBJECTIVE: To observe therapeutic effects of the comprehensive therapy of acupuncture-moxibustion and Chinese Tuina for treatment of insomnia due to deficiency of both the heart and spleen. METHODS: 92 cases were divided randomly into the treatment group (treated by acupuncture-moxibustion and Chinese Tuina) and the control group (treated by acupuncture-moxibustion). RESULTS: The therapeutic effect of the treatment group was obviously superior to that of the control group (the CHI2 test showed P < 0.01). CONCLUSIONS: The comprehensive therapy of acupuncture-moxibustion and Chinese Tuina can give marked therapeutic effects for treatment of insomnia due to deficiency of both the heart and spleen.     

Analysis of CK-19,PDX-1,Nestin,Ngn3 in different donor islet purity in rats

Objective: To investigate if there are the CK-19, PDX-1, Nestin, Ngn3 positive cells in the donor islets of different purity in rats. Methods: Thirty male adult SD rats were randomly divided into 3 groups. Islets were isolated using digestion by ductal injection of collagenase. Group Ⅰ (n=10): Separating cell preparations were not purified, Group Ⅱ(n=10): Islet sediment was purified with 25% Ficoll400 ,Group Ⅲ (n=10): Islet sediment was purified with 25% and 11% Ficoll-400. The levels of protein of CK-19, PDX-1, Nestin and Ngn3 were detected by immunohistochemistry and the mRNA of CK-19, PDX-1, Nestin, Ngn3 was amplified by RT-PCR. Results: After two different purification methods applied, three islet preparations of different purities were obtained. The difference of islet purity was significant among various groups (P＜0.05). Compared with group Ⅱ and group Ⅲ,the protein and mRNA of CK-19, PDX-1, Nestin,Ngn3 were both higher in group Ⅰ; group Ⅲ was poorly expressed. Conclusions: The three different islet purity donor islet have different CK-19, PDX-1, Nestin, Ngn3 positive cells within them, indicating that there are some islet stem cells in the purified donor islet.     

Dr.Zhang Ren''''s Experience in the Acupuncture Treatment of Different Diseases with the Same Therapeutic Principle

Dr.Zhang Ren,the chief physician,is the chairman of Shanghai Acupuncture and Moxibustion Association.Having been engaged in medicine for about 40 years,he is experienced in treating various intractable diseases.In his long years of clinical practice,he advocates taking the TCM differentiation as the basis to seek for the acupuncture method for treatment of modern intractable diseases.The author of this essay had the fortune to follow Dr.Zhang in study.The following is a summary of Dr.Zhang's experience in the acupuncture treatment for different intractable diseases with the same therapeutic principle.     

Luo Lingjie''''s Experience in Treating Chronic Nephropathy

In treating chronic nephropathy,Luo Lingjie,a chief physician,pays attention to regulating the balance between yin and yang,treating infection if present,and removing pathogenic factors.He prescribes gentle drugs and uses carefully strongly warming-tonifying ones,emphasizes the importance of persuading the patient to persist in treatment with medication and nurse one's health for recuperation,and is good at combined use of TCM and western medicine therapy and brings the merits of various therapies into full play,with obvious theraoeutic effects.     

Acupuncture Combined with Spinal Tui Na for Treatment of Primary Dysmenorrhea in 30 Cases

Objective: To observe the therapeutic effects in acupunture treatment of primary dysmenorrhea combined with spinal Tui Na, and study its mechanism. Methods: Thirty cases of the treatment group were treated by acupuncture combined with spinal Tui Na, and thirty cases in the control group were treated by routine acupuncture. Results: The total effective rate was 93.3% in the treatment group, and 73.3% in the control group, with a significant difference between the two groups (P<0.05). Conclusions: Acupuncture combined with spinal Tui Na has good prospects for treatment of primary dysmenorrhea.