混合生物人工肝细胞成分的初步研究：——乳猪肝细胞的分离 …

目的 为探讨乳猪肝细胞分离、体外培养的条件及其生长方式。方法 用两步灌注结合胶原酶消化法分离乳猪肝细胞,用激光条件培养基进行体外培养,ＥＤＴＡ消化传代,测定细胞的活性,观察其生长方式,群体倍增时间及蛋白质和ＤＮＡ的合成。结果 在 原酶浓度为０．０７５％,细胞的收获笔成活率较高,原代和传代的肝细胞生长良好,原代肝细胞倍增时间较短,ＤＮＡ和蛋白质的合成也较活跃,与传代细胞相比,无明显差别（Ｐ〉０．０５     

乳猪肝细胞的分离培养及其功能

目的探讨乳猪肝细胞分离和培养的条件及其功能．方法用门静脉插管原位灌注结合胶原酶消化法分离乳猪肝细胞,应用条件培养基对其进行体外培养,观察其形态、增殖。分化和功能等方面的变化．结果发现乳猪肝细胞的得率为（７．２±２．２）×１０９／肝,活率为９５．０％±３．０％,体外培养的乳猪肝细胞,在长达９０ｄ的培养时间内能较好地维持其形态,原代和传代培养的乳猪肝细胞其ＤＮＡ和蛋白质的合成及ＡＬＴ和Ｇ—６—ＰＤ的活性相比,虽有变化,但趋势相同,两者无统计意义（Ｐ＞０．０５）．结论体外培养的乳猪肝细胞,在一定的培养条件下,能维持其生物学特性,可作为混合型生物人工肝的细胞成分     

大量分离乳猪肝细胞的方法

目的　探讨大量分离乳猪肝细胞的方法．方法　用体外灌流装置,ＥＤＴＡ 和胶原酶两步灌流法消化分离乳猪肝细胞,综合方法观察判断肝细胞产量、活力、纯度及增殖能力．结果　分离肝细胞的总产量达到１０９／ 肝,细胞活力９４ ％ ～９８ ％ ,纯度大于９６ ％ ． 分离乳猪肝细胞胞膜完整、细胞器丰富,培养肝细胞有活跃的增殖能力,培养上清中乳酸脱氢酶浓度仅有轻度升高,２４ ｈ 平均浓度１７－８ Ｕ／Ｌ±５－４ Ｕ／Ｌ．结论　采用体外灌流装置两步分离法可获得大量高活性的乳猪肝细胞     

小鼠羊膜干细胞的分离与培养

目的探索小鼠羊膜细胞(mAMCs)分离、培养方法及其体外增殖分化的特性。方法应用不同浓度胰蛋白酶及与胶原酶按不同的先后顺序作用小鼠羊膜,并采用不同的细胞接种浓度培养细胞,分别考核最佳的分离及体外培养方案。免疫荧光及免疫组化法鉴定mAMCs的干细胞标志。结果胰蛋白酶消化及与胶原酶合用顺序不同,导致收获上皮细胞与间质细胞的数量和比例明显不同,先用胰蛋白酶消化将得到较多的上皮细胞,反之先用胶原酶消化将得到较多的间质细胞。以不同浓度接种上皮和间质细胞,原代培养2 w后生长曲线可见原代培养的mAMCs在y轴为对数坐标时呈长尾s型曲线。原代培养的上皮和间质细胞有较长的停滞期(2～4 d),指数增长期的细胞倍增时间间质细胞相对上皮细胞短,因此融合期时间间质细胞明显较短。接种浓度对间质细胞的生长曲线影响不大,但对上皮细胞有较大影响。在Chang medium C营养液中原代附壁培养mAMCs OCT3/4及SSEA-1阳性。结论应用0.25%胰蛋白酶及与0.075%胶原酶作用小鼠羊膜,可获得分离的mAMCs,原代培养mAMCs干细胞标志OCT3/4及SSEA-1阳性。     

人胎肝细胞培养上清对大鼠肝细胞增殖作用的初步观察

目的观察培养人胎肝细胞分泌上清（ＦＨＣＳ）对培养大鼠肝细胞的影响。方法用相差显微镜动态观察培养大鼠肝细胞的形态变化，并用放免分析法检测其ＤＮＡ合成量。结果ＦＨＣＳ对鼠肝细胞的原代培养有明显作用，表现为大鼠肝细胞增殖活跃、生长旺盛，维持正常形态及存活时间延长，肝细胞ＤＮＡ合成量明显增加（Ｐ＜０．０１）。结论ＦＨＣＳ对体外大鼠肝细胞有明显的增殖刺激作用，该作用可能与分离、培养胎肝细胞过程中分泌产生的多种细胞因子和营养物质有关，并为早期胎肝细胞悬液治疗重型肝炎的可能机理。     

原代猪肝细胞无血清培养

目的研究原代猪肝细胞无血清培养及肝细胞的功能。方法采用EGTA和胶原酶P两步法经肝静脉逆行灌注分离乳猪肝细胞,在无血清培养基中培养,并对不同培养时间的肝细胞生化合成及生物转化功能进行检测。结果肝细胞产量为（1．5±0．1）×10^10／肝,活率为（90．3±1．5）％,接种培养后肝细胞增生旺盛,LDH漏出第2天达到高峰,然后逐渐下降并稳定于一定水平。随着时间的延长及肝细胞数量的增加,自蛋白合成及利多卡因转化率逐渐增加,呈时间及肝细胞数量的依赖关系。结论采用本法分离获取的猪肝细胞产率高、活性强、具有良好的生物合成及生物转化功能,可作为生物人工肝较理想的肝细胞来源。     

肝细胞体外生长调控和肝细胞功能

自从分散肝组织培养肝细胞的方法于16年前建立以来,许多学者对原代肝细胞的体外培养,尤其是对如何建立适合肝细胞体外生长的条件进行了深入研究.但迄今为止,如何使原代培养中的肝细胞不失去原有的功能,并保留对各种激素的反应,仍是一个悬而未决的问题.肝脏的几类细胞中,起主要功能作用的是肝实质细胞,占肝脏细胞总数的70%.原代培养的第一步就是将这些实质细胞分离出来.Berry等于1969年首创的胶原酶灌洗肝脏的方法使这项工作获得成功.用该方法获取的肝细胞几乎为纯的实质细胞,不仅数量多,而且保留了肝细胞的各种功能.70年代初期,Bissell报道了体外研究肝功能和激素反应的所谓"最     

小鼠原代肝细胞的分离与培养

目的探求一种简易、经济的小鼠原代肝细胞的分离与培养方法。方法采用非灌注法分离小鼠肝脏,利用0．2％Ⅳ型胶原酶对肝脏消化以获取肝细胞,以DMEM培养基对肝细胞进行单层培养。结果光镜下可见培养的肝细胞呈三角形、圆形、类圆形或扁平不规则多角形,排列整齐,细胞界限清晰,胞浆丰富透亮,细胞中有核,有单核、双核是圆形或椭圆形,核仁清晰。结论此方法较适合一般实验室开展小鼠原代肝细胞的分离与培养,为进一步的实验研究提供了基础。     

机械分离与胰酶消化分离对长期培养的神经干细胞神经发生能力的影响

目的 探讨神经干细胞(NSCs)培养的分离传代技术及其在长期培养条件下的神经发生能力.方法取孕14d的Wistar胎鼠大脑皮层,消化分离后,以1×105/ml浓度进行NSCs原代培养.分别采用机械分离与胰酶消化法进行NSCs传代培养,台盼蓝活细胞计数测定分离后细胞存活率,计算细胞倍增时间和细胞增殖率.取第4、8、12、20、30代机械分离传代培养的NSCs球接种到24孔培养板中的盖玻片上,加入分化培养液,观察长期培养条件下NSCs的神经发生能力的变化.结果与胰酶消化法进行的NSCs球传代相比,机械分离的NSCs球为小细胞球和单细胞传代,传代后细胞存活率高,细胞倍增时间短,增殖能力强(P均<0.05).体外长期培养条件下,随着培养时间的延长,NSCs分化为神经元的比例逐渐降低,星形胶质细胞的比例逐渐增高;在4～12代NSCs分化为神经元的能力表现出下降的趋势,但仍较为稳定;12代后,神经元的分化比例迅速下降,星形胶质细胞的比例迅速上升.结论与胰酶消化法相比,逐步减小吸管口径的机械分离传代法,减少了传代对细胞的损伤,保证了大部分细胞间连接的完整性,显著增强了NSCs的增殖能力.体外长期扩增的NSCs,由于微环境和(或)自身基因的调控.随着传代的延续,其神经发生能力逐渐降低,分化为神经元的比例逐渐减少,分化为星形胶质细胞的比例逐渐增加.     

原代培养大鼠肝细胞分离方法比较研究

目的:探索原代培养大鼠肝细胞的最佳分离方法。方法:对比观察不同的肝细胞分离技术的培养效果。培养前,以4％台盼蓝染色判定肝细胞活性,培养48h后,观察肝细胞贴壁及生长状况。结果:①灌流消化法优于剪切消化法;②在多种灌流消化法中,经门静脉灌流消化法优于经胆总管灌流消化法及经腹主动脉灌流消化法;③在门静脉灌流的基础上,0.1％胶原酶Ⅰ37℃热消化10～15分钟,效果最好,而0.25％胰蛋白酶消化10～12分钟次之,用含EDTA的D-Hank液直接灌流分离法效果极差。结论:原代培养肝细胞的得率及活性与不同分离法包括灌流途径、灌流液、消化时间等有关。     

胰岛素瘤的解剖分布特点分析

目的胰岛素瘤是最常见的胰腺神经内分泌肿瘤，因其临床表现多样，导致诊断困难。影像学诊断尤其是超声内镜(EUS)在胰岛素瘤的诊断中起着重要作用，拥有较高的敏感性和特异性。本研究拟通过明确胰岛素瘤的解剖分布特点，以期有助于提高影像学的诊断准确率和降低漏诊率，尤其是在教育和培训实践中对于EUS的学习者更具有指导价值。 方法回顾性分析解放军总医院第一医学中心病案资料数据库1993年1月至2019年11月经外科手术、病理确诊为胰岛素瘤的患者的临床资料，检索方法采取搜索术后病理诊断为"胰岛素瘤"的病例，通过查阅病例的方法，提取出胰岛素瘤的大小和解剖分布等数据，进一步分析其特点。 结果共检索到确诊为胰岛素瘤的患者116例，其中，男45例、女71例，年龄13～76岁，平均年龄(44.4±14.85)岁。胰岛素瘤单发110例(94.8%)、多发6例(5.2%)。位置分布：头颈部46例(39.7%)，单发45例、多发1例；体尾部68例(58.6%)，单发65例、多发3例；全胰腺多发2例(1.7%)。病变大小特点：最大径0.4～3.4 cm，平均大小(1.53±0.58)cm。≤1 cm 29例、>1 cm而≤1.5 cm41例、>1.5 cm而≤2.0 cm28例，≤3 cm 15例，>3 cm 3例。年龄与肿瘤的大小相关，≤44岁患者肿瘤平均大小为(1.36±0.51)cm、>44岁患者肿瘤平均大小为(1.70±0.60)cm，P<0.05。头颈部的肿瘤大于体尾部的肿瘤，头颈部肿瘤平均大小(1.66±0.63)cm，体尾部(1.42±0.52)cm，P<0.05。 结论胰岛素瘤在胰腺体尾部较头颈部更好发；绝大多数单发，但可以全胰腺多发；多数小于1.5 cm，肿瘤的大小与患者年龄和肿瘤的解剖分布相关。     

Pathogenesis of carcinoma of the papilla of Vater

Most adenomas and carcinomas of the small intestine and extrahepatic bile ducts arise in the region of the papilla of Vater. In familial adenomatous polyposis (FAP) it is the main location for carcinomas after proctocolectomy. In many cases symptoms due to stenosis lead to diagnosis at an early tumor stage. In about 80%, curative intended resection is possible. Operability is the most relevant prognostic factor. Most ampullary carcinomas resp. carcinomas of the papilla of Vater develop from adenomatous or flat dysplastic precursor lesions. They can be sited in the ampulloduodenal part of the papilla of Vater, which is lined by intestinal mucosa. They also can develop in deeper parts of the ampulla, which are lined by pancreaticobiliary duct mucosa. Intestinal-type adenocarcinoma and pancreaticobiliary-type adenocarcinoma represent the main histological types of ampullary carcinoma. Furthermore, there exist unusual types and undifferentiated carcinomas. Many carcinomas of intestinal type express the immunohistochemical marker profile of intestinal mucosa (keratin 7?, keratin 20+, MUC2+). Carcinomas of pancreaticobiliary type usually show the immunohistochemical profile of pancreaticobiliary duct mucosa (keratin 7+, keratin 20?, MUC2?). Even poorly differentiated carcinomas, as well as unusual histological types, may conserve the marker profile of the mucosa they developed from. These findings underline the concept of histogenetically different carcinomas of the papilla of Vater which develop either from intestinal- or from pancreaticobiliary-type mucosa of the papilla of Vater. Molecular alterations in ampullary carcinomas are similar to those of colorectal as well as pancreatic carcinomas, although they appear at different frequencies. In future studies, molecular alterations in ampullary carcinomas should be correlated closely with the different histologic tumor types. Consequently, the histologic classification should reflect the histogenesis of ampullary tumors from the two different types of papillary mucosa.     

Demonstration of the mechanism of action of biguanides

Summary Palmitic acid oxidation in rat diaphragm homogenate is depressed by biguanide concentrations that are still incapable of inhibiting oxidative phosphorylation. Glucose oxidation is not directly effected by the same biguanide concentrations: however, the inhibitory effect of palmitic acid on glucose oxidation is partly removed by biguanides. Inhibition of fatty acid oxidation, which accounts for most of the metabolic effects caused by these drugs, can be regarded as the fundamental mechanism of action of biguanides. There is some evidence suggesting that these drugs might interact with carnitine, thus preventing long-chain fatty acids from being transported across the mitochondrial membrane to the site of oxidation. Traduzione a cura degli AA.     

Lack of correlation of degree of villous atrophy with severity of clinical presentation of coeliac disease

BACKGROUND AND AIM: Both the clinical presentation and the degree of mucosal damage in coeliac disease vary greatly. In view of conflicting information as to whether the mode of presentation correlates with the degree of villous atrophy, we reviewed a large cohort of patients with coeliac disease. PATIENTS AND METHODS: We correlated mode of presentation (classical, diarrhoea predominant or atypical/silent) with histology of duodenal biopsies and examined their trends over time. RESULTS: The cohort consisted of 499 adults, mean age 44.1 years, 68% females. The majority had silent coeliac disease (56%) and total villous atrophy (65%). There was no correlation of mode of presentation with the degree of villous atrophy (p=0.25). Sixty-eight percent of females and 58% of males had a severe villous atrophy (p=0.052). There was a significant trend over time for a greater proportion of patients presenting as atypical/silent coeliac disease and having partial villous atrophy, though the majority still had total villous atrophy. CONCLUSIONS: Among our patients the degree of villous atrophy in duodenal biopsies did not correlate with the mode of presentation, indicating that factors other than the degree of villous atrophy must account for diarrhoea in coeliac disease.     

血吸虫童虫生长发育及其机制的研究进展

血吸虫童虫是宿主免疫系统攻击的重要靶标,包括皮肤型、肺型和肝门型童虫。宿主分子对童虫生长发育具有重要作用。童虫生长发育机制包括免疫调节、信号转导、性别发育及凋亡等。肌动蛋白、组织蛋白酶、烯醇化酶和葡萄糖基转移酶等分子为血吸虫童虫生长发育的重要分子。本文对血吸虫童虫生长发育及其机制的研究进展做一综述。     

124例耐多药结核分枝杆菌基因突变特征分析

目的对临床分离的耐多药结核分枝杆菌相关基因的突变特征进行分析。方法对124例耐多药结核分枝杆菌以及50株敏感株的耐药相关基因(包括异烟肼inh A、kat G、oxyR-ahp C间隔区以及利福平rpo B)进行序列测定,分析其基因突变情况。结果异烟肼耐药inh A基因突变率为14.5%;kat G基因突变率为70.2%(87/124),主要位于315位;oxyR-ahp C间隔区突变率为15.3%;inh A、kat G两种基因同时突变率75.0%,三种基因同时突变率为89.5%。利福平rpo B基因突变的检出率高达95.2%,突变主要发生在531、526、516位点。结论我省耐多药菌异烟肼耐药相关基因最常见突变为kat G 315、inh A C-T(-15)、axyR-ahp C间隔区(-10)C-T,利福平为rpo B531、526、516。结合MDR-TB耐药相关基因的特征分析,可以建立一种快速、准确、特异的适合于我省的检测结核菌耐多药性的新方法。     

氯硝柳胺悬浮剂的毒性评价

目的评价氯硝柳胺悬浮剂的毒性，为现场大规模应用灭螺提供依据。方法按照中华人民共和国国家标准GB 15670-1995《农药登记毒理学试验方法》和鱼类毒性试验方法进行。结果经口、经皮肤的LDso雌、雄性大鼠均>5 000 mg/kg，经呼吸道的LCso雌、雄性大鼠均>5 000mg/m3，该药经口、经皮肤、经呼吸道毒性均属微毒类药物；兔眼用药后，观察期内无不良反应，对眼无刺激性；皮肤用药后对皮肤无刺激性。与氯硝柳胺原药、氯硝柳胺乙醇胺盐原药和氯硝柳胺乙醇胺盐可湿性粉剂相比，氯硝柳胺悬浮剂对鱼急性毒性最低。结论氯硝柳胺悬浮剂属微毒类药物，对鱼的毒性低于其乙醇胺盐可湿性粉剂，适合于现场应用。     

Assessment of quality of life of parents of children with juvenile chronic arthritis

The aim of the study was to assess the quality of life (QOL) and the psychological status of parents of children with juvenile chronic arthritis (JCA). The QOL, anxiety and depression of the parents of 28 children with JCA were evaluated and compared to those of the parents of 28 healthy children. Mothers of JCA children and mothers of healthy children reported similar QOL. The reported anxiety and depression levels were similar for mothers and fathers in both groups. The parents of children with pauciarticular-type JCA reported lower QOL and higher levels of anxiety and depression than the parents of children with other types, namely polyarticular and systemic JCA. These findings may be explained by the fact that the pauciarticular patients had shorter disease duration and were less frequently seen in the outpatient clinic. The QOL of mothers of children with JCA was found to be slightly impaired in the group of children with pauciarticular JCA. Future larger studies are needed to confirm these results, as the number of subjects in the three groups was rather low. Received: 26 September 2001 / Accepted: 8 February 2002     

Numerical Simulation of the Effect of Rate of Change of Glucose on Measurement Error of Continuous Glucose Monitors

Background

A 5-day in-patient study designed to assess the accuracy of the FreeStyle Navigator® Continuous Glucose Monitoring System revealed that the level of accuracy of the continuous sensor measurements was dependent on the rate of glucose change. When the absolute rate of change was less than 1 mg•dl⁻¹•min⁻¹ (75% of the time), the median absolute relative difference (ARD) was 8.5%, with 85% of all points falling within the A zone of the Clarke error grid. When the absolute rate of change was greater than 2 mg•dl⁻¹•min⁻¹ (8% of the time), the median ARD was 17.5%, with 59% of all points falling within the Clarke A zone.

Method

Numerical simulations were performed to investigate effects of the rate of change of glucose on sensor measurement error. This approach enabled physiologically relevant distributions of glucose values to be reordered to explore the effect of different glucose rate-of-change distributions on apparent sensor accuracy.

Results

The physiological lag between blood and interstitial fluid glucose levels is sufficient to account for the observed difference in sensor accuracy between periods of stable glucose and periods of rapidly changing glucose.

Conclusions

The role of physiological lag on the apparent decrease in sensor accuracy at high glucose rates of change has implications for clinical study design, regulatory review of continuous glucose sensors, and development of performance standards for this new technology. This work demonstrates the difficulty in comparing accuracy measures between different clinical studies and highlights the need for studies to include both relevant glucose distributions and relevant glucose rate-of-change distributions.     

治疗高血压药物的经济学评价

重视高血压治疗中的经济学评价,对利用我国有限的卫生资源来遏制高血压对人民群众的危害有着重要的现实意义。药物经济学对于药物治疗的成本和治疗的结果给予同样的关注。因为治疗高血压的费用,不仅涉及药物价格,还包括患者的危险水平,降压疗效和对临床终点事件的影响,以及治疗的依从性和安全性。因此药物经济学更强调整体成本和价-效比。低危病人,若非药价低廉,治疗的价-效比不够理想。而在高危的患者,价-效比越小越经济而不是药费越便宜越好。