GDF-5基因活化基质体内转染修复兔退变椎间盘的实验研究

目的通过GDF5基因活化基质体内转染兔退变椎间盘,观察其修复效果。方法脂质体介导的pcDNA3.1(+)/GDF-5重组质粒混入Ⅰ型胶原支架,置入兔退变椎间盘;实验分为GAM组,普通支架组,脂质体对照组,2个月后RT-PCR和Western blot观察椎间盘髓核细胞GDF-5基因和蛋白表达的稳定性,间苯三酚法检测髓核蛋白多糖含量,流式细胞仪检测细胞凋亡率来观察修复效果。结果 RT-PCR结果显示术后2个月GDF-5基因稳定表达。GAM组蛋白多糖含量高于另外两组(0.05),空白支架组和对照组之间没有差异(=0.601)。GAM组髓核细胞凋亡率明显低于另外两组(0.01),空白支架组和对照组之间没有差异(=0.902)。结论利用GAM可以有效的进行GDF-5基因体内转染并表达,从而修复退变椎间盘。     

人骨形成蛋白7基因转染骨髓间充质干细胞对兔退变椎问盘蛋白多糖的影响

目的:观察腺病毒介导人骨形成蛋白7基因(Human bone morphogenetic protein-7,hBMP-7)转染骨髓间充质干细胞(Bone mesenchymal stem cell,BMSC)后移植兔退变椎间盘.对退变椎间盘髓核组织中蛋白多糖含量的影响.方法:20只新西兰大白兔手术建立椎间盘L3/4、L4/5、L5/6退变模型,随机分为两组,每组10只:实验组L3/4、L4/5、L5/6椎间盘内注射腺病毒介导hBMP-7基因转染的BMSC;对照组L3/4、L4/5、L5/6椎间盘注射磷酸盐缓冲液(PBS).术后连续行磁共振观测椎问盘变化;12周后取材,应用RT-PCR、免疫组化检测hBMP-7的表达情况.分光光度法测量蛋白多糖含量.结果:与对照组相比,实验组的磁共振T2信号强度以及蛋白多糖含量均明显增高(P<0.05).hBMP-7的mRNA和蛋白水平在实验组中的表达水平也最高.结论:腺病毒介导hBMP-7转染BMSC后,移植退变椎间盘,可使髓核组织中蛋白多糖的含量增加.     

椎间融合引起相邻节段退变的动物实验模型

背景:直立大鼠椎间盘退变模型虽然能证明椎间融合后相邻节段椎间盘退变加重,但无法检验脊椎非融合技术是否更有优势。目的:在已确认的Beagle犬椎间盘退变模型基础上行椎间融合,了解椎间融合后是否加重相邻节段椎间盘退变。方法:将12只Beagle犬随机分为2组,对照组行椎间盘穿刺,损伤L_(5/6)椎间盘;实验组行椎间盘穿刺,损伤L_(5/6)椎间盘,1个月后行L_(4/5)椎间融合。融合后3,6个月行腰椎MRI检查,取L_(5/6)椎间盘标本进行组织学观察与PCR检测。结果与结论:(1)MRI检查:实验组融合后3,6个月出现不同程度的椎间盘突出征象,对照组未出现明显椎间盘突出征;(2)组织学观察:融合后6个月,实验组椎间盘纤维环与髓核组织结构紊乱,褶皱出现空隙,髓核细胞数量减少;对照组椎间盘髓核与纤维环分界清晰,胶原稀疏,排列有序,细胞数量较多,纤维环结构接近正常,胶原纤维排列整齐致密。实验组融合后3,6个月的Ⅱ型胶原阳性细胞少于对照组(P<0.05),且实验组融合后6个月时的Ⅱ型胶原阳性细胞少于融合后3个月时(P<0.05);(3)PCR检测:实验组融合后3,6个月的骨形态发生蛋白15、基质金属蛋白酶组织抑制因子1基因表达量高于对照组(P<0.05),且实验组融合后6个月时的骨形态发生蛋白15、基质金属蛋白酶组织抑制因子1基因表达量高于融合后3个月时(P<0.05);(4)结果表明:椎间融合后能引起相邻节段椎间盘退变的加重。     

骨形态发生蛋白2基因转染软骨细胞注入腰椎间盘损伤模型:10周测量蛋白多糖和胶原含量

背景:研究表明骨形态发生蛋白2基因转染软骨细胞可促进体外培养的髓核细胞合成细胞外基质,但其对体内退变椎间盘组织代谢的影响却罕见报道。目的:探讨骨形态发生蛋白2基因转染细胞移植治疗椎间盘退变的可行性。方法:用新西兰大白兔建立腰椎间盘损伤动物模型,建模后随机分为细胞移植组和对照组。细胞移植组注入骨形态发生蛋白2基因转染的软骨细胞,对照组注入等剂量生理盐水,两组动物在注射后2,4,6,8和10周分别取出退变的椎间盘髓核组织,并测量其蛋白多糖及胶原含量。结果与结论:与对照组比较,细胞移植组髓核蛋白多糖及胶原含量均有明显增高(P0.05)。结果提示椎间盘退变的标志之一可能为髓核内细胞外基质含量的减少,而骨形态发生蛋白2基因转染细胞移植的方法可促进细胞外基质合成,改善退变髓核的生物活性,延缓椎间盘的退变。     

腺病毒介导hTGF-β1基因体内转染兔椎间盘对髓核细胞I型、II型胶原的影响

目的:观察腺病毒载体介导外源性人转化生长因子(Ad/CMV-hTGF-β1)对兔椎间盘髓核细胞Ⅰ型、Ⅱ型胶原的影响。方法:纯种成年新西兰大白兔20只,随机分为实验组和对照组,每组10只。于实验组兔腰3、4椎间盘髓核内注射Ad/CMV-hTGF-β1(6×106pfu),注射量为20μl,对照组则注射20!μl磷酸盐缓冲液(PBS)。术后3周取出椎间盘,免疫组织化学方法观察椎间盘髓核细胞中Ⅰ型、Ⅱ型胶原的表达情况,并用VIDAS图像分析系统进行半定量分析。结果:免疫组织化学染色显示:实验组椎间盘髓核细胞Ⅰ型胶原的表达比对照组低;而Ⅱ型胶原的表达则比对照组高,差别有统计学意义(P<0.05)。结论:体内转染腺病毒介导的hTGF-β1基因能降低椎间盘髓核细胞Ⅰ型胶原的表达、提高Ⅱ型胶原的表达,提示hTGF-β1可以抑制椎间盘的退变。     

人退变椎间盘组织的基因表达谱

目的：研究人类退变椎间盘的基因表达谱，分析人退变椎间盘基因表达水平的变化。方法：按条件优化的一步法抽提人退变及正常椎间盘组织的总RNA各3例，分别用cy3，cy5荧光标记，获得2组椎间盘cDNA的探针，与含有4096条人类全长基因的cDNA表达谱芯片杂交，扫描芯片荧光信号图像，对所获得的基因进行生物信息学分析。结果：在4096条基因中，有差异表达的基因706条，358条基因表达量明显下降，298条基因表达量明显上升。在有差异表达的基因中，细胞凋亡相关类蛋白8条，其中上皮细胞膜蛋白(EMP-1)基因的表达上调明显。结论：退变椎间盘的基因表达发生变化，细胞凋亡增加可能是椎间盘退变发生和发展的因素之一。     

间断直立位下兔椎间盘退变模型的影像及病理表现*

背景：已有大量的动物模型用于研究椎间盘退变,但是尚缺乏一种符合人类椎间盘退变规律的动物模型。 目的：探讨直立位下构建椎间盘退变模型的可行性及造模的简便、易行性。 方法：新西兰大白兔42只,随机数字表法均分为实验组和对照组。将兔子固定在PVC管中,实验组将PVC管直立,对照组将PVC管平放。 结果与结论：4周时实验组和对照组间椎间盘高度指数、MRI及纤维环改变差异无显著性意义(P > 0.05),髓核PAS染色后灰度值明显增加(P < 0.05)。8,12周实验组和对照组椎间盘高度指数相比差异有显著性意义,退变椎间盘T2加权像信号明显降低,苏木精-伊红染色显示纤维环排列紊乱,PAS染色显示退变椎间盘髓核的灰度值明显变大(P < 0.05)。说明通过强迫直立位下因自身身体重力负荷对椎间盘产生影响可以成功建立椎间盘退变模型。     

GDF-5基因体外转染骨髓间充质干细胞移植与GAM体内转染对椎间盘退行性变作用的动物实验研究

目的通过对比体外生长分化因子5(GDF-5)转染并移植和基因活化基质(GAM)体内转染对椎间盘退行性变的修复作用,评价这两种方法的差异,以期为临床应用提供依据。方法日本大耳白兔32只,体质量约1.5~2.0kg,雌雄不限。取兔骨髓,分离骨髓间充质干细胞(BMSC),用脂质体介导的pcDNA3.1(+)/GDF-5基因重组质粒转染BMSC。将兔随机分为A组(正常组)、B组(转染细胞组)、C组(GAM组)、D组(对照组)。B、C、D组从L1-2、L2-3、L3-4、L4-5抽吸约湿质量5mg的髓核组织,制作椎间盘退行性变动物模型;B组植入已转染GDF-5的BMSC,C组植入GDF-5GAM,D组植入空白培养液。术后2个月将包括软骨终板在内的各组完整的椎间盘取出,采用间苯三酚法测量椎间盘髓核中蛋白多糖含量,采用流式细胞仪检测分析髓核细胞凋亡率。结果转染48 h后BMSC生长良好,体积略增大。经测定pcDNA3.1(+)/GDF-5基因转染成功后髓核内蛋白多糖含量,B组(0.2169±0.0264)与A组(0.2401±0.0300)间差异无统计学意义(P=0.149);其余组(C组、D组分别为0.1667±0.0278、0.1153±0.033 7)两两比较,差异都有显著的统计学意义(P0.01)。髓核细胞凋亡率,A组(24.55%±2.681%)与B组(30.980%±2.462%)间差异具有统计学意义(P=0.012),其余各组(C组、D组分别为38.220%±2.524%、57.530%±2.349%)两两相比,差异都有显著统计学意义(P0.01)。结论相对于利用GAM进行体内转染,体外转染BMSC然后再移植入体内的方法修复椎间盘退行性变的效率更高。     

腺病毒介导hTGF-β1基因体内转染兔椎间盘对髓核细胞Ⅰ型、Ⅱ型胶原的影响

目的观察腺病毒载体介导外源性人转化生长因子(Ad/CMV-hTGF-β1)对兔椎间盘髓核细胞Ⅰ型、Ⅱ型胶原的影响.方法纯种成年新西兰大白兔20只,随机分为实验组和对照组,每组10只.于实验组兔腰3、4椎间盘髓核内注射Ad/CMV-hTGF-β1(6×106pfu),注射量为20μl,对照组则注射20μl磷酸盐缓冲液(PBS).术后3周取出椎间盘,免疫组织化学方法观察椎间盘髓核细胞中Ⅰ型、Ⅱ型胶原的表达情况,并用VIDAS图像分析系统进行半定量分析.结果免疫组织化学染色显示实验组椎间盘髓核细胞Ⅰ型胶原的表达比对照组低;而Ⅱ型胶原的表达则比对照组高,差别有统计学意义(P＜0.05).结论体内转染腺病毒介导的hTGF-β1基因能降低椎间盘髓核细胞Ⅰ型胶原的表达、提高Ⅱ型胶原的表达,提示hTGF-β1可以抑制椎间盘的退变.     

间充质干细胞外泌体上调Mir-21调控HO-1机制促进椎间盘退变的修复

目的探讨间充质干细胞外泌体上调Mir-21调控血红素氧合酶(HO-1)机制促进椎间盘退变的修复研究。方法新西兰健康大白兔36只随机数字法分成3组,Blank组(正常大白兔)、Model组(椎间盘退变大白兔)和Mir-21组(椎间盘退变大白兔+间充质干细胞外泌体),通过建模处理、外泌体提存、免疫组织化学染色、Rt-PCR检测、HE染色、流式细胞术检测,观察外泌体形态、对比分析HO-1蛋白阳性率在椎间盘退变中的表达,Mir-21、HO-1和BACH1在大白兔椎间盘组织中的表达,大白兔椎间盘组织形态学变化,白兔退变髓核细胞凋亡能力情况。结果 Blank组HO-1蛋白阳性率为(62.23±4.1)%,细胞颗粒呈棕黄色,Model组的蛋白阳性率为(8.96±2.4)%,明显低于Blank组(P0.05),Mir-21组蛋白阳性率为(42.38±3.46)%,细胞颗粒呈淡黄色,显著高于Model组低于Blank组(P0.05);Model组的Mir-21、HO-1表达水平明显低于Blank组和Mir-21组(P0.05),Mir-21组的Mir-21、HO-1表达水平显著低于Blank组(P0.05)。Model组的BACH1水平明显高于Blank组和Mir-21组(P0.05),Mir-21组的BACH1水平显著高于Blank组(P0.05);Blank组兔髓核内基质比较高,髓核细胞颜色较深,周边胞浆颜色较淡,Model组通过建模后髓核细胞消失,与Blank组相比基质密度比较低,髓核细胞难见(P0.05),Mir-21组通过Mir-21转染后,与Model组相比髓核内基质明显升高,密度染色加深,髓核细胞出现,并且染色加深(P0.05);Model组和Mir-21组的细胞凋亡能力明显低于Blank组(P0.05),Mir-21组细胞凋亡能力显著高于Model组(P0.05)。结论间充质干细胞外泌体上调Mir-21,抑制BACH1来促进HO-1机制修复椎间盘退变的能力,对退变组织细胞具有促凋亡能力。     

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.     

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.     

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.     

The universal muscular chain reaction,muscle spasm,Torsions, ruptures and extravasations. Chameleons of pathology and some manifestations of simple muscular disorders

Most bodily functions require the coordinated actions of complementary and supplementary paired muscle groups. Where this essential muscular cooperation is lacking, hollow organs may burst and others become literally screwed up, giving rise to many similar spastic diseases such as Torticollis, Twisted ovarian cyst, Torsion of the Testis, Volvulus of the intestines, Varicose Veins, Megacolon, Aortamegaly, Scoliosis, Erb's Palsy, Peyronie's Disease, Main-en-Griffe, Undescended Foot (Pes Cavus), Talipes, Strabismus. Spasm is “panenepidemic” and unclassified examples of Torsion Dystonia and Dyskinesia really are as common as debt and taxes.     

Class II HLA in Georgia Caucasus Tbilisi Georgians and their Mediterranean ancestry: The Usko Mediterranean languages

Georgia (or Sakartvelo in its own language) is a South Caucasus Mts. country with its easternmost part is enigmatically named Iberia, like the Iberian Peninsula, which may refer to rivers “Kura” and “Ebro” or their valleys respectively. Most of their inhabitants speak Georgian which is included within Dene-Caucasian group and Usko-Mediterranean subgroup of languages. The latter includes Basque, Berber, ancient Iberian-Tartessian, Etruscan, Hittite, Minoan Lineal A and others. In the present paper, HLA class II -DRB1 and -DQB1 alleles has been studied and extended haplotypes calculated. Most frequent haplotypes are also of Mediterranean origin (i. e.: (A*02-B*51)-DRB1*11:01-DQB1*03:01, (A*02-B*51)-DRB1*13:01-DQB1*06:03, or (A*24-B*35)-DRB1*01:01-DQB1*05:01) and DA genetic distances show that closest world populations to Georgians are Mediterraneans. Georgians also show common extended haplotypes ((A*02-B*51)-DRB1*11:01-DQB1*03:01, (A*02-B*13)-DRB1*07:01-DQB1*02:01 and (A*03-B*35)-DRB1*11:01-DQB1*03:01) with Svan people, a secluded population in North Georgia mountains. We can conclude that Georgians belong to a very old Mediterranean substratum according to both linguistics (Usko Mediterranean languages) and HLA genetics.     

'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.     

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.