改良Nash差速贴壁联合阿糖胞苷法体外分离培养大鼠嗅球及嗅黏膜源性嗅鞘细胞*☆

背景：目前常用的嗅鞘细胞培养方法有差速贴壁法、化学抑制法、抗体亲和吸附法、补体法等,各自均存在优缺点,单一使用某种方法时细胞纯化率较低。 目的：拟采用改良Nash差速贴壁+阿糖胞苷法体外分离纯化大鼠嗅球及嗅黏膜来源的嗅鞘细胞。 设计、时间及地点：细胞学体外对照观察,于2007-11/2008-05在武汉大学人民医院骨科实验室和消化内科实验室完成。 材料：10周龄Sprague-Dauley大鼠10只,由武汉大学人民医院实验动物中心提供,阿糖胞苷由武汉大学人民医院制备。 方法：完整取大鼠双侧嗅球及剪取鼻中隔后1/3嗅黏膜,剪碎后胰酶消化,制成单细胞悬液,按1×109 L-1密度接种于未包被的25 cm2玻璃培养瓶中,18~20 h后将细胞悬液转移至另一未包被的25 cm 2玻璃培养瓶中（第1次差速贴壁）,24 h后再将细胞悬液转移至经多聚右旋赖氨酸包被的25 cm2塑料培养瓶或经多聚右旋赖氨酸包被的6孔培养板中（第2次差速贴壁）,接种培养48 h后半量换液以去除杂细胞。在差速贴壁培养后二三天,加入3.0~5.0 pmol/L阿糖胞苷去除残余成纤维细胞。 主要观察指标：嗅鞘细胞的形态观察、分裂增殖情况、免疫荧光染色鉴定结果及纯度测定。 结果：体外培养24 h嗅球源性嗅鞘细胞即可贴壁,而嗅黏膜源性嗅鞘细胞多在培养四五天后贴壁,两种来源的嗅鞘细胞形态相似,以双极或梭形细胞为主,少量为3极及多突起形多级细胞,同时夹杂扁平、煎鸡蛋形细胞。纯化培养10 d的嗅鞘细胞,胶质纤维酸性蛋白、神经生长因子受体p75免疫荧光染色及神经生长因子受体p75+hoechs免疫荧光双染后大部分双极或多极细胞膜、胞体、突起呈阳性,细胞纯度可达90%以上。 结论：改良Nash差速贴壁+阿糖胞苷法可在体外成功分离培养出高纯度的嗅球及嗅黏膜源性嗅鞘细胞,嗅球源性嗅鞘细胞贴壁时间及分裂增殖程度优于嗅黏膜源性嗅鞘细胞。     

胰酶限时消化在嗅鞘细胞培养中的运用

目的探索出一套高效、实用的大鼠嗅球嗅鞘细胞(OECs)的培养和纯化方案。方法分离SD大鼠嗅球的外两层组织,剪切消化成细胞悬液进行接种。采用3种方法进行OECs的培养和纯化:(1)A组分别经6h、24h两次差速贴壁去除杂质细胞,培养至12d左右消化重悬细胞进行爬片分析。(2)B组分别经6h、24h两次差速贴壁去除杂质细胞,培养至12d左右经胰酶限时消化,留成纤维细胞于瓶壁,重悬的细胞进行爬片分析。(3)C组采用经典的Nash法(分别经18h、36h两次差速贴壁去除杂质细胞),培养至11d左右消化重悬细胞进行爬片分析。采用NGFRP75与碘化丙啶(PI)双染的方法进行OECs的鉴定和纯度分析。结果在共聚焦显微镜下呈双染阳性的细胞为OECs,OECs多数突起细长,呈双极或三极,少量呈单极或多极。A、B、C组所纯化的OECs纯度分别为(67.3±6.2)%、(83.7±7.7)%和(74.6±9.5)%,3组间比较有统计学差异(F=13.633,P<0.01),B组所得OECs纯度均较另两组高(P<0.05)。结论经6h、24h两次差速贴壁+胰酶限时消化可以获得较高纯度的OECs,能够满足动物实验的需要。     

改良差速贴壁+无血清条件培养液纯化培养大鼠嗅鞘细胞*

背景：细胞移植是脊髓损伤的一种有效治疗手段,嗅鞘细胞被认为是最适合的种子细胞之一,但目前对其培养纯化的方法尚无公认标准。 目的：运用改良差速贴壁法+含同源嗅鞘上清液的无血清条件培养液来纯化培养嗅鞘细胞,以期建立一种新颖、经济、简单、实用的纯化培养成年大鼠嗅鞘细胞的方法。 设计、时间和地点：观察性对照实验。实验于2008-02/06在苏州大学干细胞与组织工程实验室完成。 材料：健康成年SD大鼠6只,体质量150~180 ｇ。 方法：①无菌条件取大鼠嗅球组织,消化、离心去除上清液后,用含体积分数为0.2胎牛血清的DMEM/F-12培养基重悬,稀释的单细胞混悬液均分成3份,分别用于单纯改良差速贴壁、单纯无血清条件培养液纯化和改良差速贴壁+无血清条件培养液纯化的实验。②单纯改良差速贴壁：将单细胞混悬液以8 000个/cm²密度种植于无包被的25 cm²培养瓶中。经12 h+ 24 h两次差速培养后,吸出细胞上悬液,计数板计算浓度后,按1×109 Ｌ-1浓度重新种植于载有多聚赖氨酸包被盖玻片的35 mm培养皿中继续培养3周。③单纯无血清条件培养液纯化：将单细胞混悬液用预先收集并制备的含同源嗅鞘培养上清液的无血清条件培养液直接种植于载有多聚赖氨酸包被盖玻片的35 mm培养皿中,孵育两三天后再换成含体积分数为0.1胎牛血清DMEM/F12培养基继续培养3周。④改良差速贴壁+无血清条件培养液纯化：经12 h +24 h两次差速纯化,吸出上悬液接种于培养皿中继续培养2.0~3.0 d后,改换成无血清条件培养液,孵育36~48 h后再换成含体积分数为0.1胎牛血清DMEM/F-12培养基继续培养。以后视情况每3~5 d半量换液1次,培养3周。 主要观察指标：运用倒置显微镜观察各组不同时间的嗅鞘细胞生长状况和形态学特征。采用GFAP、NGFRp75和Hoechst33258等抗体,于分离培养14 d后进行细胞免疫荧光染色并检测嗅鞘细胞的纯度。 结果：①倒置显微镜观察：差速后3 d内有大量细胞贴壁生长,细胞形态较混杂,辨认嗅鞘细胞较困难。差速后再运用无血清条件培养液2 d后,可见纯度较高的典型嗅鞘细胞形态,以双极梭形和多极为主,细胞突起细长。伴少量扁平状“油煎蛋样”细胞。继续培养至14 d可见细胞立体感及折光性最佳,数量和纯度最高。培养3周后3组细胞开始老化,活力明显下降,成纤维等杂细胞开始挤占原嗅鞘细胞生长空间。②免疫荧光染色显示嗅鞘细胞特异性表达GFAP和NGFRp75,单纯差速后嗅鞘细胞纯度仅有72%~75%,单纯无血清条件培养液纯化后仅为48%~52%,改良差速贴壁+无血清条件培养液纯化组纯度可达88%~92%。 结论：实验建立了完善的成年大鼠嗅鞘细胞培养纯化新方法。     

牛垂体提取物、碱性成纤维细胞生长因子及Forskolin影响嗅鞘细胞增殖的量效关系

背景：生长因子参与调节嗅鞘细胞增殖、分化及代谢功能,因此有望通过应用其合理的序贯刺激形式,达到细胞数量地大量扩增。 目的：观察牛垂体提取物、碱性成纤维细胞生长因子及Forskolin对嗅鞘细胞增殖的影响及剂量-效应关系。 方法：采用原代培养技术,从新生大鼠的嗅球分离培养嗅鞘细胞,并用差速贴壁+化学药物+胰酶快速消化法纯化培养嗅鞘细胞,添加牛垂体提取物、碱性成纤维细胞生长因子及Forskolin 3种促增殖因子及其组合。MTT法检测各组嗅鞘细胞生长的情况。根据NGFRp75抗体的免疫细胞化学染色统计嗅鞘细胞的纯度和计算嗅鞘细胞的增殖率。 结果与结论：在嗅鞘细胞原代培养和纯化后,添加牛垂体提取物具有促进嗅鞘细胞增殖的效应,但未见典型的剂量-效应关系;添加碱性成纤维细胞生长因子具有促进嗅鞘细胞增殖的效应,且呈现典型的剂量-效应关系;添加Forskolin并不具有促进嗅鞘细胞增殖的效应。说明合理应用不同质量浓度的碱性成纤维细胞生长因子与Forskolin进行联合刺激,能有效促进细胞的增殖。结果表明序贯的使用合适浓度的生长因子组合,将是有效促进嗅鞘细胞增殖的合理策略,有助于解决脊髓损伤治疗中种子细胞来源不足这一问题。     

分离、培养及纯化嗅鞘细胞的简便方法

目的：建立一种免胰酶消化的剪切分散法提取嗅鞘细胞（olfactory ensheathing cells,OECs）并优化其纯化培养方法。方法：从成年SD大鼠解剖分离嗅球,显微镜下剥离软脑膜、剔除中央白质,将灰质成分置于含10%胎牛血清的DMEM/F12全培养基,眼科剪剪切至组织成糜状,吸管吹打15-20次, 混悬液免胰酶消化直接接种培养,每三天半量换液。原代培养至细胞大部分融合后,依次以低浓度胰酶（0.05%）有限消化法、30min短时差速贴壁法、抗有丝分裂法三步顺序纯化细胞,每步纯化结果作免疫荧光染色鉴定。结果: 原代培养中嗅鞘细胞快速贴壁成为优势细胞、生长旺盛,三步顺序纯化过程细胞纯度逐步提高,最终纯度可达95%以上并长时间维持。结论:剪切分散法提取OECs及其三步顺序法纯化培养经济实用、易重复,细胞培养周期短,能收获大量可控纯度的嗅鞘细胞供进一步移植实验及基础研究。     

用于移植的嗅鞘细胞的纯化培养方法研究

目的 探讨移植用成年大鼠嗅球嗅鞘细胞(OECs)的培养和纯化方法.方法 取成年SD大鼠嗅球,将分离消化所得细胞悬液进行接种.然后分别于接种后2 h、6 h和18 h 3个时间点进行单步骤差速贴壁法去除成纤维细胞,纯化嗅鞘细胞,纯化培养约11 d时采用神经生长因子受体P75(NGFRP75)和碘化丙啶(PI)双染OECs并对其纯度及细胞数量进行免疫荧光鉴定分析.结果 纯化培养11 d后在共聚焦显微镜下呈双染阳性的细胞为OECs,数量级达106/mL,形态以梭形、双极或三极突起为主,亦有少量多极或单极细胞.2 h、6 h和18 h组嗅鞘细胞纯度分别为(53±5)%、(73±8)%和(69±13)%,组间差异有统计学意义(F=11.766,P<0.001). 结论分别经2、6、18 h单步骤差速贴壁法纯化的OECs在纯度和数量级上均可满足移植用细胞的要求,且方法简单、经济、快捷.     

改良成年大鼠嗅鞘细胞的原代培养

目的研究成年大鼠嗅鞘细胞(OECs)分离与培养的方法,为进一步进行基础研究以及临床应用提供高纯度的细胞.方法采用改良差速贴壁法从成年大鼠的嗅球培养出OECs,并用Forskolin和碱性成纤维细胞生长因子(bFGF)促增殖,最后进行OECs特异标记物NGFRp75、S-100的免疫细胞化学染色鉴定OECs.结果改良差速贴壁法成功培养出高纯度的OECs.结论此法简便、经济、纯化效率高,Forskolin和bFGF能够促进OECs的增殖.     

大鼠嗅球嗅鞘细胞的体外分离培养及纯化

背景：差速贴壁+Thy1.1抗体及补体纯化法的应用较多,细胞换液过程中使用含血清的培养基主要针对成纤维细胞的去除,而对神经元的去除作用不明显。 目的：根据神经元体外培养需要血清的特性,拟利用无血清培养基在体外建立一种分离培养嗅鞘细胞的简单方法。 设计、时间及地点：细胞学体外观察,于2008-06/2009-01在哈尔滨医科大学动物实验中心完成。 材料：成年SD大鼠10只,由哈尔滨医科大学实验动物中心提供。 方法：在差速贴壁+Thy1.1抗体及补体纯化法的基础上,剪开大鼠颅骨,显露位于颅腔前方的嗅球,取出2只嗅球,在显微镜下去除嗅球表面的软脑膜和毛细血管及外周组织,保留富含嗅鞘细胞的嗅神经层和嗅球颗粒层,剪成1 mm3小块分离获取单细胞悬浮液,调整细胞密度至1×107 L-1,接种在用poly-l-lysine包被的培养瓶或培养板中,于37 ℃、体积分数为5%的CO2培养箱中培养,第3天用无血清DMEM/F12培养基换液培养。 主要观察指标：嗅鞘细胞的形态学观察、生长曲线分析、免疫荧光染色结果。 结果：培养前3 d细胞数目无明显增加,甚至减少;然后细胞数目逐渐增多,13 d细胞基本长满;传10代后细胞形态发生明显变化,胞浆内出现许多空泡,细胞增殖速度减慢或停止。纯化培养10 d后,培养板内双极和三极细胞均呈GFAP,NGFR p75阳性。 结论：在差速贴壁+Thy1.1抗体及补体纯化法的基础上,利用无血清DMEM/F12培养基进行换液培养可使嗅鞘细胞迅速增殖,是一种效率较高的体外培养方法。     

酶消化及差速贴壁法原代培养新生C57小鼠耳蜗膜蜗管外侧壁成纤维细胞

背景：目前对耳蜗膜蜗管外侧壁成纤维细胞的培养没有特定的方法。 目的：采用酶消化与差速贴壁相结合的方法,原代培养新生C57小鼠膜蜗管外侧壁成纤维细胞,提供良好的体外细胞模型。 方法：显微解剖分离新生C57小鼠膜蜗管外侧壁组织,对膜蜗管外侧壁组织进行胰蛋白酶消化与差速贴壁相结合的方法培养成纤维细胞,倒置相差显微镜下观察细胞生长状态,苏木精-伊红染色,绘制细胞生长曲线,免疫组织化学染色鉴别细胞来源。 结果与结论：膜蜗管外侧壁组织来源传代纯化的成纤维细胞呈梭形和三角形,生长曲线成S形,免疫组织化学检测波形蛋白,细胞胞浆中呈棕黄色阳性反应。结果证实培养出小鼠膜蜗管外侧壁成纤维细胞     

预损伤嗅黏膜对自体嗅鞘细胞体外培养的作用

目的：比较预损伤嗅黏膜与正常嗅黏膜来源的嗅鞘细胞生长特性和生物学活性的差异。 方法：雄性成年SD大鼠10只，随机分为嗅黏膜损伤组、正常组，5只/组。嗅黏膜损伤组大鼠麻醉后采用自制弯头针，深入鼻腔内直至鼻中隔后1/3处，针尖抵住鼻中隔后切割数下至出血，填塞酒精棉条压迫止血。正常组大鼠不做任何处理。伤后7 d取两组大鼠鼻中隔，将双侧鼻黏膜后1/3从鼻中隔刮下，胰酶消化后体外分离培养嗅黏膜嗅鞘细胞，调整浓度至1×109 L-1，采用改良NASH差速贴壁法进行纯化。 结果：嗅鞘细胞纯度达70%时，正常组需14 d，嗅黏膜损伤组需12 d。正常组培养7 d后细胞进入对数期，嗅黏膜损伤组培养5 d后细胞进入对数期，两组嗅鞘细胞进入平台期后生长趋于缓慢，数量维持在106左右。在细胞生长周期的相同时间点，嗅黏膜损伤组嗅鞘细胞活性大于正常组（t=19.001 3，P < 0.001），两组嗅鞘细胞分泌的神经营养因子3质量浓度基本相似 （P > 0.05）。 结论：预损伤处理嗅黏膜能够加快嗅黏膜嗅鞘细胞的增殖分化，且活性较高。     

Pathological attachment and attachment style in late adolescence

Attachment theory is frequently invoked to account for patterns of adaptation within relationships. West and Sheldon derived a measure of dysfunctional adult attachment from Bowlby's theory. Four patterns are identified: compulsive self-reliance; caregiving; care-seeking; and angry withdrawal. The aim of this study was to assess the psychometric properties of this promising measure, and to assess its ability to predict symptomatology relative to measures of attachment style. Participants included 209 late adolescents who were involved in committed relationships. They responded to categorical and continuous measures of attachment style and various measures of symptomatology and college adjustment. The four dimensions of pathological attachment showed strong internal consistency and few gender differences. Compulsive care-seeking, angry withdrawal and compulsive self-reliance were particularly strong predictors of psychiatric symptomatology and insecure attachment style. Attachment pathology and insecure attachment style predicted symptomatology similarly, though variations were evident depending on how style was assessed. This study is the first to provide evidence of convergent, discriminant and predictive validity for this measure. Directions for future research are noted.     

The therapist's attachment representation and the patient's attachment to the therapist

Abstract

This study examines the relationship between the therapist's own attachment representation (Adult Attachment Interview, AAI) and the patient's attachment relationship to the therapist (client's attachment to the therapist, CATS). The attachment representations of n=22 psychotherapists who treated n=429 patients were assessed. A general effect of the secure/insecure therapist attachment status on the attachment of the patient to the therapist was not found whereas the more specific effects expected could be confirmed: The more preoccupied the therapist's attachment status was, the more the patient experienced a preoccupied-merger attachment to the therapist. The more dismissing the therapist's attachment status was, the more the patient experienced an avoidant-fearful attachment to the therapist. In summary, not the general security/insecurity of the therapist's attachment representation but rather the type of the insecurity is associated with the subjective patient's attachment-related experience of the therapeutic dyad.     

Adult attachment styles and psychosis: an investigation of associations between general attachment styles and attachment relationships with specific others

BACKGROUND: If attachment theory is to help inform our understanding of relationship difficulties in people with psychosis, it is first important to understand the composition of attachment networks in this group and how attachment style measured with reference to relationships in general actually relates to attachment in relationships with specific others. METHOD: We examined attachment networks and associations between general attachment style and attachment in relationships with parents and psychiatric staff in a sample of 58 patients with psychosis. We assessed attachment dimensions of anxiety and avoidance using the Psychosis Attachment Measure (PAM) and an adapted version of the instrument asking about relationships with specific others. RESULTS: Patients reported a median of two attachment relationships. Both attachment anxiety and avoidance measured with reference to close relationships in general were positively correlated with attachment in key worker and parental relationships, although levels of attachment anxiety and avoidance did vary across relationships. CONCLUSION: Future research should determine factors influencing variations in attachment working models in samples of people with psychosis, as it may be possible to help individuals with insecure attachment styles develop more positive relationships with others.     

Volunteer client attachment moderates the relationship between trainee therapist attachment and therapist interventions

Abstract

This study explored the association between attachment security of therapists (alone and in interaction with clients' attachment) and therapist interventions in early sessions of short-term psychotherapy. Trainee therapists and volunteer clients (N=24) in short-term therapy completed the Experiences in Close Relationship Scale (Brennan, Clark, & Shaver, 1998) as a measure of adult romantic attachment orientations. Therapist interventions were identified and related to client and therapist attachment orientation. Results indicated that in early therapy sessions client attachment moderated the relationship between therapist attachment and therapist interventions. Specifically, avoidantly attached therapists intervened with more directive interventions when clients were high in attachment avoidance.     

Depression and attachment problems.

This study investigated the characteristics related to attachment of 42 depressed psychiatric patients and 42 non-depressed psychiatric controls. The depressed subjects demonstrated an anxious pattern of attachment, characterized by either intense care-seeking in relation to their attachment figure or angry withdrawal from their attachment figure when their desire for security was frustrated. The results are discussed in terms of Bowlby's attachment construct.