以转染CDMP1基因的BMSCs修复软骨缺损

 目的 探讨CDMP1基因转染的骨髓间充质干细胞（BMSCs）负载于聚乳酸-羟基乙酸（PLGA）支架上修复喉软骨缺损的能力,并对其修复效果做出初步评估。方法 用反转录聚合酶链式反应（RT-PCR）和免疫印迹法(Western blot)检测hCDMP1mRNA和蛋白的表达;用免疫组织化学方法检测Ⅱ型胶原蛋白(ColⅡ)以及糖胺聚糖(GAG)的表达;将转染前后的细胞支架培养体系移植入兔甲状软骨全层缺损处,从大体、组织学方面观察其对软骨缺损的修复作用。结果 腺病毒感染方法可以将外源hCDMP1基因成功转入BMSCs,并使其获得稳定表达;和对照组比较,转染hCDMP1基因的BMSCs分泌ColⅡ、GAG等软骨特异性基质的能力增强,有促进软骨分化趋势;转染细胞支架复合物可更加有效地修复喉软骨缺损。结论 转染hCDMP1基因的BMSCs/PLGA三维生物支架复合物移植动物体内可修复喉软骨缺损。     

骨形态发生蛋白质-4转染骨髓间充质干细胞复合双层聚乳酸羟基乙酸共聚物支架修复兔膝关节软骨缺损

目的:探讨骨形态发生蛋白质-4(BMP-4)基因转染骨髓间充质干细胞(BMSCs)复合聚乳酸羟基乙酸共聚物(PLGA)修复兔全层关节软骨缺损的效果。方法:取兔骨髓间充质干细胞,分离培养后,免疫组织化学鉴定BMSCs。采用电转法将pcDNA 3.1-BMP-4质粒导入BMSCs。转染成功后培养备用。制备直径为4 mm的双层PLGA支架。新西兰大白兔随机分成空白组、PLGA组、PLGA/BMSCs组、PLGA/BMP-4/BMSCs组4组,将双层PLGA支架置入兔双膝关节股骨内侧髁软骨缺损中。观察并记录术后动物膝关节活动情况。第8、16周时取膝关节置入组织行H-E染色,在扫描电镜下观察置入的PLO认新生组织。采用RT-PCR法定量检测4组修复软骨缺损处新生的软骨Ⅱ型胶原、SOX-9、蛋白聚糖基因的表达水平。结果:术后各组动物膝关节活动正常,未见炎症反应。第8、16周时PLGA/BMP--4/BMSCs组兔膝关节损伤修复优于其他3组。H-E染色结果可见PLGA/BMP-4/BMSCs组膝关节损伤修复效果优于其他3组。RT-PCR结果显示PLGA/BMP-4/BMSCs组兔置入的PLGA新生的软骨中Ⅱ型胶原、SOX--9、蛋白聚糖基因的表达水平显著高于其他各组。结论:BMP--4转染BMSCs后能够促进骨髓间充质干细胞分化成软骨细胞,双层PLGA支架置入有利于兔膝关节软骨缺损区的修复。     

功能性纳米自组装多肽KLPP凝胶修复软骨缺损的实验研究

目的 探讨功能性纳米自组装多肽KLPP凝胶对关节软骨缺损的修复作用。方法 培养人骨髓间充质干细胞（BMSCs）,将BMSCs分为BMSCs-KLD-12组（BMSCs与KLD-12凝胶共培养）和BMSCs-KLPP组（BMSCs与KLPP凝胶共培养）,分别行软骨诱导分化培养,MTT法检测细胞的增殖活性。将人软骨组织分为软骨-KLPP组（制作软骨缺损区并填充KLPP凝胶进行培养）、软骨-KLD-12组（制作软骨缺损区并填充KLD-12凝胶进行培养）、软骨组（仅制作软骨缺损区）。Calcein-AM/PI双染法检测BMSCsKLD-12组及BMSCs-KLPP组的细胞毒性。培养49 d后,收集软骨组、软骨-KLD-12组、软骨-KLPP组的软骨组织进行切片,并行阿尔新蓝染色。Western blot法检测各组中Ⅰ型胶原（Col-Ⅰ）、Ⅱ型胶原（Col-Ⅱ）、Ⅹ型胶原（Col-Ⅹ）的表达。结果 与BMSCs-KLD-12组比较,BMSCs-KLPP组培养1 d、3 d、7 d的细胞增殖率均明显增加（P<0.05）,且Col-Ⅰ、Col-Ⅱ、Col-Ⅹ的相对表达水平明显增加（P<0...     

不同来源蚕丝蛋白修复骨软骨组织缺损的效果比较

背景：目前还没有研究比较不同种属来源蚕丝蛋白修复骨软骨的效果。 目的：观察桑蚕和柞蚕来源蚕丝蛋白支架材料修复骨软骨缺损的效果差异。 方法：取新西兰兔20只,制备单侧膝关节骨软骨缺损模型,随机分为5组：其中1组不植入任何材料,作为空白对照;实验1组将3层桑蚕丝蛋白支架粘在一起,充填于缺损处;实验2组将1个包被转化生长因子β3的桑蚕丝蛋白支架与2个包被骨形态发生蛋白2的桑蚕丝蛋白支架粘在一起,充填于缺损处;实验3组将3层柞蚕丝蛋白支架粘在一起,充填于缺损处;实验4组将1个包被转化生长因子β3的柞蚕丝蛋白支架与2个包被骨形态发生蛋白2的柞蚕丝蛋白支架粘在一起,充填于缺损处。术后8周,取关节骨软骨修复区行组织病理学观察,检测Ⅰ型和Ⅱ型胶原蛋白表达。 结果与结论：实验1组胶原蛋白广泛分布于缺损区全层;实验2组胶原纤维在修复区表面呈平行分布,在中间和底部呈垂直顶部的方向分布;实验3组在修复区表面可见胶原;实验4组在支架表面和底部可见软骨样细胞有成团分布。4个实验组修复区Ⅰ型胶原蛋白呈强阳性表达;实验1组、实验2组修复区Ⅱ型胶原蛋白呈微弱表达,实验3组、实验4组修复区Ⅱ型胶原蛋白呈强阳性表达。表明两种蚕丝蛋白均能修复骨软骨缺损,桑蚕丝蛋白倾向于形成骨组织,柞蚕丝蛋白倾向于形成软骨组织。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程      

CDMP和TGF联合诱导BMSCs修复软骨缺损

目的 探讨骨髓间充质干细胞(BMSCs)在软骨源性形态发生蛋白1(CDMP1)和转化生长因子-β(TGF-β1)联合诱导前后成软骨能力的差异.方法 采用免疫组织化学和阿新蓝染色的方法,检测BMSCs与聚乳酸/羟基乙酸共聚物(PLGA)构筑的三维立体培养体系在CDMP1和TGF-β1联合诱导前后,产生特异性软骨基质Ⅱ型胶...     

腺病毒携带骨形态发生蛋白14基因转染脂肪干细胞修复损伤关节软骨

背景：关节软骨损伤后自我修复能力较弱,主要是由于其缺乏滋养血管并且细胞代谢缓慢等组织特性,目前的治疗方法都不能恢复软骨组织的原有功能,近年来软骨组织工程已引起了越来越多的关注。 目的：观察Ⅰ型胶原海绵支架搭载骨形态发生蛋白14基因转染脂肪干细胞修复兔膝关节软骨损伤的效果。 方法：取兔皮下脂肪组织分离培养脂肪干细胞,用腺病毒真核表达载体Ad-CMV-BMP-14-IRES-hrGFP-1转染脂肪干细胞。Ⅰ型胶原海绵支架搭载转染后的脂肪干细胞,待细胞吸附后对兔膝关节全层软骨缺损进行修复。术后12周取手术关节,从大体方面、组织学方面综合评估缺损修复状况。 结果与结论：骨形态发生蛋白14转染后的脂肪干细胞骨形态发生蛋白14和Ⅱ型胶原蛋白表达及Sox-9基因表达明显高于普通脂肪干细胞。术后12周,支架搭载经骨形态发生蛋白14转染的脂肪干细胞组软骨组织修复良好,平整光滑,光洁度、质地及颜色良好,交界区整合良好。支架搭载脂肪干细胞组软骨组织部分修复,有正常软骨光泽,质地与颜色接近正常,修复组织与正常软骨组织界限明显。单纯支架组几乎崩解塌陷,未见透明样软骨结构形成。结果可见腺病毒携带骨形态发生蛋白14基因转染后脂肪干细胞修复软骨缺损的能力有大幅提升。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

凝胶包埋骨髓基质干细胞复合脱钙骨基质修复关节软骨缺损

目的探索Ⅱ型胶原凝胶包埋的自体骨髓基质干细胞(BMSCs)接于同种异体脱钙骨基质(DBM)材料修复兔关节软骨缺损的效果。方法15只健康成年新西兰大白兔,雌雄不限,体质量约3.0 kg,兔龄6~9个月;以Urist方法制作同种异体DBM材料。以Ⅱ型胶原蛋白配制水凝胶,以水凝胶包埋兔BMSCs并接种于同种异体DBM材料,构建组织工程复合物。在新西兰大白兔股骨髁关节面制造软骨缺损,分组进行修复。将健康成年新西兰大白兔27只(雌雄不限,体质量约2.5 kg,兔龄3~4个月)共54侧膝关节随机分为Ⅱ型胶原/DBM/BMSCs修复组(实验组)、Ⅱ型胶原/DBM修复组(实验对照组)及空白对照组。于术后4周、8周及12周各处死9只动物,取材对修复组织进行大体及组织学观察,根据Wakitani法对修复组织进行评分,数据输入SPSS 11.5软件进行统计学分析,比较各组的评分差异是否具有统计学意义。结果实验组Ⅱ型胶原/DBM/BMSCs植入后形成透明软骨样修复,表面光滑平坦,与周围软骨及软骨下骨结合良好;实验对照组Ⅱ型胶原/DBM植入后有部分软骨样修复;而空白对照组仅有少量纤维性修复。根据组织学评分标准,实验组组织学评分为(20.25±1.64)分,高于实验对照组[(7.46±1.29)分]及空白对照组[(6.00±2.09)分]。结论Ⅱ型胶原自体BMSCs复合同种异体DBM支架材料修复全层关节软骨缺损的效果良好,是一种修复软骨缺损的行之有效的方法。     

软骨下钻孔联合软骨源性形态发生蛋白缓释系统修复膝关节软骨缺损

背景：有研究表明软骨源性形态发生蛋白等因子在诱导细胞分化、促进软骨修复过程中起到重要调节作用;软骨下钻孔治疗软骨缺损在临床已广为应用,但其与软骨源性形态发生蛋白等因子联合应用的相关研究至今少有报道。 目的：将软骨下钻孔技术与关节内注射透明质酸/软骨源性形态发生蛋白1缓释载药微球(hyaluronic acid- coated cartilage-derived morphogenetic protein-1,HA/CDMP-1)相结合,观察其对软骨缺损修复的效果,并通过与单纯钻孔组及HA/CDMP-1微球组治疗结果比较,观察两者有无协同效应。 方法：用透明质酸包被软骨源性形态发生蛋白制备缓释微球冻干保存,制备兔实验膝关节全层关节软骨缺损模型,随后将兔随机分为模型组、钻孔组、HA/CDMP-1微球组和钻孔联合HA/CDMP-1微球组,分别采用生理盐水关节腔注射,软骨缺损区钻孔,HA/CDMP-1微球关节腔注射,软骨下钻孔并HA/CDMP-1微球注射。 结果与结论：建模后8,12,16周,组织学观察结果提示,钻孔联合HA/CDMP-1微球组软骨缺损区修复组织覆盖面积明显高于其他3组(P < 0.05);甲苯胺蓝染色和荧光定量PCR检测显示,钻孔联合HA/CDMP-1微球组修复的软骨组织中蛋白多糖和Ⅱ型胶原mRNA的表达明显高于其他3组(P < 0.01或P < 0.05)。结果证实,软骨下钻孔与关节腔内注射HA/CDMP-1联合应用修复兔膝关节软骨缺损近期疗效满意,提示两者可能发生协同作用。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

GDF-5和Dex通过Wnt/β-catenin信号通路促进rBMSCs体外成软骨分化

目的利用生长分化因子(GDF-5)联合地塞米松(Dex)诱导大鼠BMSCs成软骨分化,并探讨Wnt/β-catenin信号通路在其中的作用。方法用全骨髓贴壁法分离培养SD大鼠BMSCs,取P3代细胞随机分成6组:BMSCs组、BMSCs+Dex组、BMSCs+GDF-5组、BMSCs+GDF-5+Dex组、BMSCs+GDF-5+Dex+SB216763组和BMSCs+GDF-5+Dex+XAV-939组。连续诱导培养14 d,倒置相差显微镜观察细胞形态,Alcian blue染色检测细胞的蛋白聚糖,RT-PCR检测成软骨分化相关基因aggrecan、Col2、Sox9、Col1,Wnt/β-catenin信号通路相关基因Dvl1、Gsk3β、β-catenin mRNA表达,Western blot检测Ⅱ型胶原蛋白和β-catenin蛋白表达。结果与BMSCs组相比,BMSCs+GDF-5+Dex组能够使诱导的细胞蛋白聚糖分泌明显增加;Aggrecan、Col2、Sox9基因表达明显增加(P0.05);Ⅱ型胶原蛋白表达增加(P0.05);β-catenin mRNA及蛋白表达水平也增加(P0.05)。与BMSCs+GDF-5+Dex组相比,添加SB216763组蛋白聚糖,Ⅱ型胶原基因和蛋白表达增加(P0.05);相反,添加XAV-939组相应的成软骨分化基因及蛋白表达下降(P0.05)。结论 GDF-5联合地塞米松(Dex)可诱导大鼠BMSCs成软骨分化,Wnt/β-catenin信号通路可能在其中发挥促进作用。     

组织工程支架的隔离层对新生软骨Ⅱ型胶原蛋白表达的影响

目的研究隔离层在组织工程骨软骨复合支架中,对新生软骨Ⅱ型胶原蛋白表达的影响。方法将向软骨方向诱导后的兔骨髓间充质干细胞接种到骨软骨复合支架的软骨支架上,然后将具有隔离层和没有隔离层的骨软骨复合支架分别植入兔膝关节骨软骨全层缺损处。术后3月和6月分别取材,进行新生软骨Ⅱ型胶原蛋白蛋白的免疫组化染色、Western blot法检测Ⅱ型胶原蛋白的表达、实时荧光定量PCR(qRT-PCR)测定Ⅱ型胶原蛋白相关基因的表达。结果与无隔离层的对照组相比,具有隔离层的骨软骨复合支架所形成的新生软骨的Ⅱ型胶原蛋白和基因的表达均明显增强(P<0.05)。结论隔离层加入骨软骨复合支架后,能显著提高新生组织工程软骨Ⅱ型胶原蛋白表达的能力。     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.