抗溴化脱氧尿嘧啶核苷单克隆抗体免疫荧光细胞化学染色过程的最佳条件实验研究

抗溴化脱氧尿嘧啶核苷(BrdUrd)单克隆抗体荧光染色过程涉及到以下一些重要步骤:BrdUrd脉冲标记时间长短,核DNA部分变性处理所用盐酸的浓度与处理时间等。应以BrdUrd阳性细胞与阴性细胞之间平均荧光强度之比值,酸处理过程中细胞凝集的比例,细胞群体G_1峰DNA荧光强度及变异系数作为整个染色过程最佳条件的评价标准。在30分钟BrdUrd标记时间,2.4mol/L盐酸30分钟处理时间及抗BrdUrd单克隆抗体20℃1小时温育时间等条件下,可获得满意的染色结果,并且不需要核糖核酸酶的处理。本实验室将KF-1、KFr、HeLa、IK-90等几种培养细胞系用此方法染色均可获得满意的染色结果。     

Assessment of the influence of subacute phenobarbitone administration on multi-tissue cell proliferation in the rat using bromodeoxyuridine immunocytochemistry

The effects of daily administration of phenobarbitone on the mitotic rates of several tissues were investigated by bromodeoxyuridine (BrdU) immunocytochemistry. Phenobarbitone (80 mg/kg per day) was dosed to AP Wistar male rats for up to 7 days and BrdU (10 mg/ml) was given by infusion at a rate of 10 l/h via subcutaneously implanted osmotic minipumps for 2 days prior to necropsy on days 1, 2, 3, 5 and 7. BrdU-labelled nuclei were visualised by peroxidase-antiperoxidase immunocytochemistry and counts of the numbers of labelled cells (labelling index, LI%) made from at least 1000 cells per tissue section(s). The LIs of several tissues (testis, adrenal cortex and medulla, kidney distal convoluted tubule and exocrine pancreas) showed no statistical difference by comparison with controls. Several tissues exhibited characteristic responses to phenobarbitone administration. Pituitary and endocrine pancreas LIs were decreased while those of thyroid, liver and kidney proximal convoluted tubule were increased. The pattern of LI increase was unique to each tissue with liver (median and lateral lobes) increased two-fold on day 3 and returning to control levels thereafter while kidney proximal tubule LI rose gradually with time and remained elevated on day 7. Thyroid LI on day 1 was almost double that of day 0 control and increased steadily thereafter. These data illustrate the varied responses of different tissues to phenobarbitone exposure, namely, depression and stimulation of mitosis. The causation of these functional changes is discussed in relation to direct and indirect effects on functional parameters, especially enzyme induction, alterations in hormonal and growth factor status and receptor regulation.     

Rapid detection of S-phase cells by anti-bromodeoxyuridine monoclonal antibody in 9L brain tumor cells in vitro and in situ

Summary FITC-conjugated anti-bromodeoxyuridine (BrdUrd) monoclonal antibody (anti-BU-MAb) was used to detect S-phase cells of 9L rat brain tumor cells in vitro and in situ. Monolayer 9L cells were treated with 0.625–20 M of BrdUrd for 30 min, harvested, and reacted with a 1:100 dilution of FITC-conjugated anti-BU-MAb and analyzed with a flow cytometer. BrdUrd-treated cells stained satisfactorily with antibody. Values obtained for the labeling index using this method (48.6%) were 10%–20% higher than the fraction of cells in S-phase calculated from DNA histograms or as the labeling index calculated from autoradiographs of cells pulse-labeled with³H-thymidine. BrdUrd (1–40 mg/kg) was administered by i.p. injection to rats bearing 9L brain tumors. Single cell suspensions obtained by disaggregation of excised tumors were stained with anti-BU-MAb. The percent-age of fluorescent cells (15.9%) calculated using this method was similar to that of S-phase cells (17.2%) calculated from DNA histograms and from autoradiographics for tumor bearing rats pulse-labeled with³H-thymidine in situ. The antibody staining technique is a rapid and accurate method for various cell kinetic studies both in vitro and in vivo in a rat model, and has promise as a technique for the study of cell kinetics in humans.Supported in part by grants PDT159 from the American Cancer Society, CA-13525 from National Cancer Institute and the gift from Aaron Silvera Cancer Research Fund     

Cell Proliferation in Colorectal Adenocarcinomas: Comparison Between Ki-67 Immunostaining and Bromodeoxyuridine Uptake Detected by Immunohistochemistry and Flow Cytometry

We compared three different means of assaying tumor proliferative activity in 30 human colorectal adenocarcinomas labeled in vivo with bromodeoxyuridine (BrdUrd). The labeling indices (LI) of BrdUrd obtained both by flow cytometry (FCM) and immunohistochemistry (IH) were also compared with the labeling index of Ki-67. These methods were then related to tumor ploidy and pathological features. Flow cytometry was performed in accordance with Begg's method after intravenous infusion of BrdUrd four hours before surgery. Immunohistology was carried out on paraffin-embedded sections with monoclonal antibodies against BrdUrd and Ki-67. A positive correlation was found between BrdUrd LI obtained by both FMC and IH (p<0.0001), a finding that complies with the literature. However, we report on a correlation between Ki-67 LI and BrdUrd LIs in colorectal tumors (p=0.012). The results were valid for all tumors when they were subdivided into diploid and aneuploid groups. The labeling indices were significantly higher in the aneuploid tumor group than in the diploid group (p=0.047). No relationship between proliferation parameters and tumor stage or grade was found. To our knowledge, this is the first report on a positive correlation between tumor proliferation indices in BrdUrd LIs and Ki-67 in colorectal carcinomas. This finding validates the value of Ki-67 immunostaining, which, however, should be confirmed in a larger series under the same technical conditions.     

Colectomy with ileorectal anastomosis lowers rectal mucosal cell proliferation in familial adenomatous polyposis

Rectal polyp regression has been observed in familial adenomatous polyposis (FAP) after colectomy and ileorectal anastomosis (IRA). In view of the association between risk of neoplasia and epithelial cell turnover rates, a study was conducted to determine the effect of colectomy and IRA on rectal mucosal proliferation in FAP. Endoscopic biopsies of flat rectal mucosa were taken from 12 FAP patients with an established IRA and 10 FAP patients prior to colectomy. Mucosal proliferation was assessed by flash-labeling S-phase cells with bromodeoxyuridine. Labeled cells were visualized on paraffin sections with an immunohistochemical stain using a monoclonal antibody to bromodeoxyuridine. Twenty crypt columns were analyzed. The mean labeling index (percent labeled cells/crypt) of the FAP patients with established IRAs (7.0±1.4 percent) was significantly less than that of the precolectomy patients (12.8±3.0 percent) (Mann-Whitney U test,P =0.0004). Comparison of labeled distribution curves shows a contraction of the crypt proliferative zone in the IRA group. Colectomy with IRA in FAP is associated with a significant reduction in rectal mucosal cell proliferation. These findings support the claim of reduced risk of rectal cancer following this procedure in FAP and are of relevance to the study of environmental vs.genetic control of cell proliferation.Presented at the meeting of the British Society of Gastroenterology, London, September 1991.Dr. Farmer was supported by the St. Mark's Research Foundation.     

Donor–host tissue interaction in allogenic transplanted tooth germ with special reference to periodontal tissue

Objectives

Limited biological evidence exists regarding donor–host interaction in the periodontal tissue during allogenic tooth germ transplantation. This study aimed to clarify donor–host tissue interactions during periodontal tissue healing following tooth germ transplantation.

补阳还五汤对缺血性脑损伤后成年大鼠海马齿状回神经发生的影响

目的 :观察补阳还五汤对缺血性再灌注脑损伤后成年大鼠海马齿状回神经发生的影响。方法 :使用四血管阻断方法 (4 VO)制作成年大鼠全脑缺血再灌注模型。采用神经前体细胞 (BrdU)标记分裂细胞方法 ,观察、比较缺血再灌注脑损伤后 3、6、1 2、2 4、36天时补阳还五汤干预组大鼠与相应对照组大鼠之间海马齿状回神经前体细胞的增殖水平。结果 :缺血再灌注脑损伤后 ,空白干预缺血组大鼠各时间点海马齿状回BrdU阳性细胞数量水平与缺血对照组大鼠相比均无显著性差异 (P >0 0 5) ;而补阳还五汤干预组大鼠齿状回BrdU阳性细胞数量较缺血对照组大鼠和空白干预组大鼠则明显增加 (P <0 0 1 )。结论 :补阳还五汤可明显促进缺血再性灌注脑损伤后海马齿状回神经发生水平的上调 ,其机制可能与补阳还五汤有效成分的多种生物学活性有关