血液细胞冷冻干燥保存的研究进展

血液细胞的冷冻干燥保存与传统的常温保存和低温保存相比，具有储存期限长、储存运输方便、输注安全等优点。本文从血液细胞冷冻干燥保存的细胞学基础，血小板、红细胞、脐带血冷冻干燥保存的现状，发展血液细胞冷冻干燥保存技术需要解决的关键问题等几个方面进行综述。     

血液细胞冷冻干燥保存的研究进展

血液细胞的冷冻干燥保存与传统的常温保存和低温保存相比,具有储存期限长、储存运输方便、输注安全等优点。本文从血液细胞冷冻干燥保存的细胞学基础,血小板、红细胞、脐带血冷冻干燥保存的现状,发展血液细胞冷冻干燥保存技术需要解决的关键问题等几个方面进行综述。     

冷冻干燥保存血小板的研究进展

血小板是血液中的一种重要组成成分,它主要参与血栓形成和凝血过程.在大量失血和化疗、放疗等处理后造成的血小板减少症患者的治疗过程中,需要进行血小板输注.因此,保证足够的血小板贮备和供应显得尤为重要.目前,临床应用血小板存在供应不足和浪费问题,主要是血小板保存技术在方法学上的局限性所造成的.血小板的保存方式有以下三种:①常温(22±2℃)保存,此条件下保存的血小板由于部分活性的丧失和微生物的污染,根据美国药品监督管理局规定最多只能保存5天;②冰冻保存,这种保存方法可以大大延长血小板的保存时间,临床实验证明冻-融血小板聚集功能仅为新鲜血小板的50%～60%,而且需要笨重的冰箱和存储设备;③冷冻干燥保存,冷冻干燥血小板可以在常温下长时间保存,不需要冷冻存储设备.在此,本文对冷冻干燥保存血小板(简称冻干血小板)进行综述.     

冷冻干燥保存血小板的实验研究

本研究探讨冷冻干燥保存血小板的方法,以获得可长期冻干保存的血小板制品,使之能在常温条件下保存、占用空间小、重量轻、便于长距离运输,能够满足突发事件和战伤救治的需要。在冷冻干燥保存过程中添加血小板可逆性激活抑制剂、DMSO和海藻糖等低温保护剂,进行预处理、冷冻、一级干燥、二级干燥,再水化,并同时测定血小板回收率,凝血酶聚集反应,促凝血功能,CD62p表达率和PAC-1表达率等。结果表明:血小板回收率为56.29%,其对凝血酶的聚集反应与对照组无明显差异,对ADP和丙基没食子酸诱导的聚集反应较对照组分别降低49.34%和26.25%,促凝血功能与对照组比较也无统计学差异;冻干血小板CD62p表达率为42.36%,PAC-1表达率为2.12%,凝血酶激活后CD62p再表达率为50.88%,PAC-1再表达率为54.55%。结论:添加血小板可逆性激活抑制剂,海藻糖和DMSO后的冻干血小板,其聚集活性和促凝血功能与新鲜血小板无明显差异,血小板可逆性激活抑制剂降低了冻干血小板的CD62p表达,增强了冻干血小板的生存能力,因而延长了其生存时间,因此可以该冻干方法为基础进一步提高冻干保存血小板的效率。     

细胞内外海藻糖对红细胞冰冻干燥保存的影响

目的研究细胞内海藻糖对红细胞冻干保存后血红蛋白回收率及ATP水平影响。在一定条件下负载红细胞,细胞内海藻糖浓度保持恒定,研究细胞外不同浓度的海藻糖对红细胞冰冻干燥保存的影响。方法将浓缩红细胞在37℃,浓度为800 mmol的海藻糖溶液中孵育7 h,制成海藻糖负载的浓缩红细胞,对照组为PBS负载浓缩红细胞,行冻干保存,测定Hb回收率及细胞内ATP水平。将PBS液、浓度为50 mmol、200 mmol、400 mmol的海藻糖溶液与海藻糖负载浓缩红细胞按1∶1比例混匀,行冻干保存及再水化,用氰化血红蛋白试剂盒测定Hb溶血率,计算Hb回收率。结果经海藻糖负载的红细胞冻干保存,Hb回收率(44.46±5.15)%,细胞内ATP水平(1.91±0.33)μmol/gHb,对照组Hb回收率(7.71±2.71)%,细胞内ATP水平(0.88±0.25)μmol/gHb。2组相比较,P0.05,差异有统计学意义。细胞外PBS液组Hb回收率为(10.36±0.97)%,50 mmol海藻糖组,Hb回收率为(33.57±2.89)%,200 mmol海藻糖组,Hb回收率为(38.64±0.54)%,400 mmol海藻糖组,Hb回收率为(18.10±1.9)%。对照组PBS液组与50 mmol组、200 mmol组、40 0mmol组分别比较,差异有统计学意义。400 mmol组与200 mmol组、5 0 mmol组分别比较,差异有统计学意义(P0.01)。200 mmol组与50 mmol组相比较,差异无统计学意义。结论细胞内的海藻糖大大提高冻干红细胞Hb回收率且可保持冻干红细胞正常ATP水平。细胞外的海藻糖对红细胞冻干保存有保护作用,随着细胞外液中海藻糖浓度增加,冻干红细胞Hb回收率减少。     

海藻糖负载红细胞及其冻干保存研究

为了研究海藻糖负载红细胞方法的可行性及红细胞内海藻糖对冻干红细胞的影响,利用红细胞膜在37℃时细胞膜上部分脂质由固态变为液态、流动性增大和膜通透性增加的性质,将红细胞置于高浓度海藻糖负载液中孵育7小时,并以磷酸缓冲盐溶液中孵育的红细胞作为对照,对红细胞的海藻糖负载率、形态学、渗透脆性、变形性、ATP含量及2,3-DPG含量进行评价。结果表明:负载后红细胞内海藻糖含量为36.56±7.95mmol/L,实验组红细胞溶血率为(15.663±3.848)%,对照组红细胞溶血率为(5.03±1.85)%,差异显著(P<0.05);实验组红细胞变形指数是0.0289±0.00738,对照组红细胞变形指数是0.1200±0.0121,差异显著(P<0.05);负载后实验组红细胞内ATP含量为2.67±0.54μmol/gHb,对照组红细胞内ATP含量为5.22±1.10μmol/gHb(P>0.05),实验组红细胞渗透脆性降低,明显低于对照组。尽管负载组的红细胞大小不一,形态各异,但在透射电镜下绝大多数红细胞膜完整,胞内血红蛋白密度均匀,而对照组有近一半的细胞膜不完整并有漏孔,胞内血红蛋白密度变浅。实验组与对照组中2,3-DPG含量均为零。实验组红细胞冻干再水化后,血红蛋白回收率46.44±4.14%,对照组血红蛋白回收率8.33±2.34%,差异显著(P<0.001)。结论:海藻糖负载的红细胞功能符合输注标准,负载方法可行,负载入细胞内的海藻糖能够保持细胞膜的完整性,大大提高了冻干红细胞的回收率,为红细胞的冷冻干燥成功迈出了第一步。     

不同冻干保护体系对海藻糖负载红细胞冻干保存影响的研究

本研究旨在评价冻干保护剂人血白蛋白、葡聚糖、聚乙烯吡咯烷酮和甘油对海藻糖负载后红细胞冰冻干燥保存的影响,筛选最佳冻干保护体系。将浓缩红细胞在37℃,浓度为800 mmol/L的海藻糖溶液中孵育7 h,经PBS液冲洗3遍后制成海藻糖负载的浓缩红细胞。对照组为海藻糖负载红细胞不添加保护剂,直接冻干;实验组将人血白蛋白、葡聚糖、聚乙烯吡咯烷酮、甘油等组成的冻干保护体系与海藻糖负载浓缩红细胞混合,两组样品在常温下平衡30 min,移入-80℃深低温冰箱,预冻24 h,入冻干机冻干处理24 h。用温度为37℃,6%羟乙基淀粉40注射液快速再水化样品,用氰化血红蛋白试剂盒测定血红蛋白溶血率,计算血红蛋白回收率,同时测定干燥样品含水量。结果表明：当样品含水量在3%-4%时,对照组冻干红细胞血红蛋白回收率为（33.57±2.89）%,白蛋白组血红蛋白回收率为（51.15±1.98）%,差异有显著性意义（P〈0.05）。选用不同浓度的葡聚糖为冻干保护剂,血红蛋白回收率较对照组明显降低,随浓度增加,血红蛋白回收率逐渐升高,当浓度为36%时,血红蛋白回收率为（22.15±4.12）%,差异有显著性意义（P〈0.05）。不同浓度的聚乙烯吡咯烷酮（PVP）组成的冻干保护体系,当浓度小于40%时,血红蛋白回收率明显低于对照组,差异有显著性意义（P〈0.05）。10%甘油组血红蛋白回收率为（3.93±1.80）%,差异有显著性意义（P〈0.05）。结论：人血白蛋白在海藻糖负载的冻干红细胞中发挥重要保护作用,葡聚糖与浓度小于40%PVP可削弱细胞内海藻糖的保护作用。液态的甘油不宜作为红细胞冰冻干燥保存的保护剂。     

冷冻干燥保存兔血小板的实验研究

目的研究冷冻干燥长期冻干保存的血小板制品的方法。方法将24只大白兔随机分为3个实验组:在所抽取的兔血小板中添加血小板可逆性激活抑制剂、DMSO和海藻糖等低温保护剂,经过预处理、冷冻、一级干燥、二级干燥等过程冷冻干燥兔血小板,分别室温保存1 d(实验1组)、7 d(实验2组)和15 d(实验3组)后经再水化;分别与各自冷冻干燥前的新鲜血小板为对照。并检测血小板回收率和聚集反应功能,并用851Cr示踪法评价血小板体内生存活性。结果3个实验组血小板冷冻干燥后回收率分别为71.68%,68.76%和70.64%,组间无明显差异。实验1、2、3组对凝血酶的最大聚集率(73.20%,77.53%和71.31%)与对照组(86.20%)差异无明显统计学意义(P>0.05),对ADP和没食子酸诱导的聚集反应率(35.6%和58.0%、36.61%和62.56%,32.65%和60.12%)比对照组低(72.4%和91.0%);冻干后保存兔血小板的1、24和48 h体内生存率分别为70%—79%、52%—56%和32%—40%,3组间差异无统计学意义(P>0.05)。结论添加血小板可逆性激活抑制剂、海藻糖和DMSO后冷冻干燥获得的冻干兔血小板,具有较好的聚集活性和体内存活率,保存活性较为稳定。     

保护液的玻璃化状态对红细胞冷冻干燥保存后回收率的影响

为了研究保护液的玻璃化状态对红细胞冷冻干燥 (简称冻干 )保存后回收率的影响 ,采用含有 7%二甲亚砜 (v/v)和 2 0 %、30 %、4 0 %或 5 0 %聚乙烯吡咯烷酮 (PVP) (w/v)的缓冲液作为保护液进行保护液的玻璃化测试和红细胞的冻干保存实验。首先检测溶液的玻璃化状态 ,如果冷冻和解冻过程中任一过程出现白色冰晶即为非玻璃化溶液 ;再将浓集红细胞和不同的保护液按比例混匀 ,预冻后移入冻干机内进行冻干处理 ;冻干完毕后 ,快速水化样品 ,测定红细胞回收率、血红蛋白回收率和上清游离血红蛋白浓度 ,然后对冻干后红细胞形态进行电镜观察。结果表明 :2 0 %PVP +7%DMSO和 30 %PVP +7%DMSO在冷冻和解冻过程中都出现白色冰晶 ;4 0 %PVP +7%DMSO在冷冻过程中无冰晶出现 ,但在解冻过程中出现冰晶 ;而 5 0 %PVP +7%DMSO在冷冻和解冻过程中均无冰晶出现。冻干红细胞再水化后 ,4 0 %PVP +7%DMSO的细胞回收率和血红蛋白回收率分别为 (81.36±14 94 ) %和 (77.5 4± 12 .86 ) % ,显著高于其它 3组 (P <0 .0 1) ;另外 4 0 %PVP +7%DMSO的上清游离血红蛋白浓度也显著低于其它 3组 (P <0 .0 1)。研究表明 :随着溶液中PVP浓度的升高 ,溶液的玻璃化程度也随之增加 ,同时冻干 再水化后红细胞的各项指标也随之改善 ,当溶液中PV     

自体脐带血的保存及临床应用

脐带血内含有丰富的造血干、祖细胞,脐血移植已成功应用于治疗白血病、淋巴瘤、骨髓异常增生综合征、再生障碍性贫血、血红蛋白病、代谢贮积病和免疫缺陷性疾病.随着脐血库的建立和临床脐血移植数量的增多,越来越多的父母选择将脐带血捐献给公共库或者是储存到自体库以备应用.本文就近年来自体脐带血保存的现状和临床应用做一综述.     

Successful preservation of human skin by vitrification

The vitrification technique was applied to the preservation of human skin. This technique was simple, and no expensive equipment was needed. Split-thickness human skins from 8 patients were immersed in vitrification solution for 10 minutes at room temperature, immediately plunged into a liquid nitrogen tank, and cryopreserved for 3 weeks. The vitrification solution consisted of 40% ethylene glycol (vol/vol) and phosphate buffered saline solution that contained 30% Ficoll 70 (vol/vol; Wako Junyaku, Co, Tokyo, Japan) and 0.5 mol/L sucrose. The viability of vitrified and cryopreserved skin was evaluated with the trypan blue dye exclusion test, the methyl-thiazoldiphenyl-tetrazolium (MTT) colorimetric assay, and a culture test of the keratinocytes obtained from vitrified skin. The results of the trypan blue dye exclusion test showed 87.4% of viable cells, and MTT developed an average 0.817 absorbance. When vitrified skin was compared with 4 degrees C refrigerated skins after 3 weeks of storage, the difference of viability was significant both on the trypan blue dye exclusion test (P < .05) and on the MTT assay (P < .01). However, there was no significant difference in the viability of vitrified skins compared with fresh skin. Furthermore, keratinocytes from vitrified skin grew uneventfully in culture test. We used these vitrified skin allografts for patients with flame burns and electric burns. These allografts took well in both cases and promoted wound healing. We concluded that the vitrification method for skin preservation is simple and reliable, and this method could contribute to skin banking.     

手术前备皮方法-剃毛与不剃毛的实验研究与临床意义

手术前清洁皮肤剃除手术野的毛发,被列为外科手术前常规,其利弊如何,已有学者提出异议,笔者做了一组临床实验证明:剃毛备皮组24h后皮肤表面细菌存留数明显高于不剃毛备皮组。剃毛备皮组每平方厘米细菌菌落数在8个以上者有17例,不剃毛备皮组仅4例。两组备皮后24h表面单位面积菌落数有显著意义(P<0.01),为此,剃毛备皮法易增加术后感染。     

Neurofibrosarcoma of skin and subcutaneous tissues.

In this report, we describe 13 cases of primary neurofibrosarcoma of the skin. The tumor presumably arises from small cutaneous nerves, is locally aggressive, and has a potential for metastasis. Characteristic histopathologic features include proliferating atypical spindle cells with slender wavy and pointed nuclei; hypocellular areas with loose, myxoid stroma; and areas of organoid organization such as palisading, whorly, storiform, and tactile body-like formations. The S-100 stain is positive in about 60% of cases. In the current series, most tumors arose in deep dermis and were grade 2 malignant lesions with a moderate degree of cytologic atypia and 2 or fewer mitoses in 10 high-power fields. Three patients died of their malignant lesion. Only two tumors metastasized. Of the 10 patients who had local recurrence, 5 had multiple recurrent lesions. Neurofibrosarcoma should be considered in the differential diagnosis of malignant tumors of the skin. A complete surgical resection of the primary tumor with adequate margins of surrounding normal-appearing tissue is advised.     

Short-term skin preservation at 4 degrees C: skin storage configuration and tissue-to-volume medium ratio

This study was designed to examine the effect of the storage configuration of skin and the ratio of tissue-to-storage medium on the viability of skin stored under refrigeration. Human skin specimens were stored in four physical configurations in RPMI 1640 tissue culture media at 4 degrees C. Skin was transferred to surgically created defects on nude mice after specific storage intervals. Grafts were examined grossly and microscopically after ten days. In the rolled configuration, on storage day 15, the viability of the outside of the roll was significantly better than the inside (P less than 0.01). The graft viability of the outside of the skin rolls was similar for both tissue-to-media ratios as well as for both free-floating configurations (P = 0.27). These findings suggest the optimum cold storage configuration is free floating, and 300 cm2/100 mL is an appropriate skin surface area to volume media ratio. This proportion of tissue to media is in agreement with the minimum ratio currently recommended by the Skin Council of the American Association of Tissue Banks.     

组织工程复合皮肤修复大鼠皮肤缺损

目的实验室培养大鼠组织工程皮肤,探讨临床可行性。方法以新生SD大鼠皮肤为原材料培养组织工程复合皮肤并将其移植到成年Wistar大鼠,通过大体观察、组织学检测对自体、异体及组织工程复合皮肤移植进行比较,研究了组织工程复合皮肤在修复皮肤缺损中的可行性。结果异体皮肤移植组有明显的免疫排斥,组织工程皮肤组未见有明显排斥,且与自体皮肤结合良好。结论组织工程复合皮肤与实验大鼠结合良好,可用于修复大鼠皮肤缺损。     

Characterization of light penetration in rat tissues

OBJECTIVE: The goal of this study is to determine the optical properties of different rat tissues with respect to spatial intensity variation and light distribution. We are interested mainly in the wavelength of 630 nm. Nevertheless, for liver tissue we have used 514 nm and 670 nm as well. BACKGROUND DATA: In the past, many articles have been written about the interaction of lasers with rat tissues. However, the technique of imaging the light distribution allows us to obtain the spatial scattering as well as an effective attenuation coefficient for the light intensity. METHODS: Slices of different rat tissues were placed between two microscope slide mounts (spaced by 3 mm). A laser beam was irradiated on the sandwiched tissue. A CCD camera placed on the side, orthogonal to the beam path, recorded the intensity distribution of the scattered light. Analysis of this spatial intensity profile allowed determining the variation of the intensity as the light penetrates the tissue. RESULTS: We have found that abdominal wall fat presents the lowest exponential decay when compared with liver, muscle, and kidney. The obtained values provided good data about the light distribution in those tissues when irradiated with a nondiffuse laser beam. For all tissues, we observed a spherical light distribution and exponential decay. Cirrhotic liver shows much stronger decay than healthy liver. These results are useful for several applications of laser for biostimulation a phototherapy.     

丹参素在大鼠主要组织分布中的动力学特征

背景:近年来对丹参素体内药动学的研究已有很多,但丹参素在大鼠主要组织中特别是皮肤和骨组织中的分布动力学研究尚未见全面的报道.目的:研究大鼠静脉注射复方丹参液后丹参素在丰要组织中的分布动力学.方法:采用液液萃取法对SD大鼠血清和主要组织样品(肾、脾、心、肝、肺、腹部皮肤、背部皮肤和骨组织),以对羟基苯甲酸为内标,甲醇-1%冰醋酸(8:92)为流动相,用高效液相色谱法于280 nm处检测,液质联用进行定性分析.结果与结论:大鼠静脉注射复方丹参液后,除脑组织外,在血清和其它组织中均检测到丹参素,用所建立的方法绘制了其经时血药浓度曲线和组织浓度柱形图.研究表明大鼠静脉注射复方丹参液后,可以在主要组织中(除脑外)快速分布,其动力学类型属二室模型.