Analysis of DNA Content of Various Types of Spermatogenic Cells in Rat after Testicular Heating with Flow Cytometry

To measure DNA contents of spermatogenic cells and analyze the efficiency of spermatogenesis after testicular heating in rat Methods Eighty adult male Sprague-Dawley rats were randomly divided into experimental group （43 ℃, 30 min） and control group （22 ℃, 30 min）. According to day 0.5, 1, 3, 6, 10, 25, 35 and 50 after local testicular heating, every group was divided into 8 subgroups： experimental subgroups （n=6） and control subgroups （n=4）. DNA contents of various types of germ cells were observed with flow cytometry （FCM） in all groups. Results Compared with control groups, percentages of 4C cell （primary spermatocyte） in 0.5-35 d groups and percentages of 1C cell （spermatid and sperm） in 6-50 d groups significantly decreased in experimental groups （P〈0.05）, and percentages of 2C cell （spermatogonium and second spermatocyte） in 3 -35 d experimental groups increased significantly after heating （P〈0.05）. 4C：2C in all of 8 experimental groups and 1C：2C in 3-35 d experimental groups were down （P〈0. 05）, and in 1 d experimental group 1C：4C was up after heating （P〈0.05）. Conclusions After being heated, the number of spermatocyte firstly decreased, and then that of spermatid and sperm decreased too. Heat influences several stages in spermatogenesis and results in suppression of spermatogenesis. Flow cytometry is an effective method for researching on the change of spermatogenesis and has significance on mechanism about changing of spermatogenic cells induced by heat.

Analysis of DNA Ploidy, Cell Cycle and Ki67 Antigen in Nasopharyngeal Carcinoma by Flow Cytometry

Summary: The expression of DNA ploidy, the cell cycle and Ki67 antigen in nasopharyngeal carcinoma (NPC) were studied and their relationship with the clinical biological behaviors and prognosis of NPC was evaluated. Biopsied specimens of NPC were made into cell suspension. By using cytometric double labeling Ki67 and DNA method, the expression of DNA ploidy, the cell cycle and Ki67 antigen were analyzed. The patients were followed-up for about 3 years and the relationship between the above-mentioned parameters and the clinical biological behavior and prognosis of NPC were evaluated. Of the 62 cases of NPC, the DNA aneuploid accounted for 29.03 %. The S phase cells accounted for 0 to 54 % in the cell cycle and the positive expression of Ki67 ranged from 0 to 52 %. There were 40 cases of LPI (64.5 %) including 15 negative cases and 22 cases of HPI (35 5 %) respectively. The DNA anueploid content was positively related to the S phase cells. The patients having a low expression of Ki67 or DNA aneuploid in tumor cells were not sensitive to chemotherapy, liable to metastasis to distant organs and had a poor prognosis, while Ki67 showed no correlation with DNA ploidy and the cell cycle. It was suggested that DNA ploidy and Ki67 could be used as an independent and objective marker to evaluate the radiosensitivity and prognosis of NPC.

Flow Cytometric Analysis of DNA Content in Parotid Tumor and Its Contiguous Acini

Summary: To investigate the relationship between proliferative capacity of salivary gland cells in contiguous acini of parotid tumors and recurrent neoplasma, DNA contents of 30 fresh specimens of parotid were studied by using cytometry in tumors, normal and shallow or deep lobe acini of the masses. The results showed that the DI was 1. 369, S % 16. 95, PI 26. 18 in malignant tumors; DI was 1. 171, S % 12. 41, PI 15. 54 in recurrent pleomorphic adenoma; DI was 1. 141, S % 12. 74, PI 13. 07 in pleornorphic adenoma, DI was 0. 999, S % 5. 10, PI 8. 00 in normal acini. Analysis of variance showed there was a significant difference (P<0. 01 ). The average DNA contents of shallow on deep lobe of contiguous tumors was 1. 08 in DI, 10. 65 in S %, 13. 49 in PI in malignant tumor, 1. 06 in DI, 8. 96 in S % and 9. 85 in PI in pleomorphic adenoma, which were all higher than in normal acini (P>0. 05). It was concluded that the levels of DI and S % of parotid tumor and its contiguous acini are related to degree of malignancy or recurrent condition of the tumors, suggesting contiguous acini of parotid tumors had the strong capacity of proliferation, which might play an important role in recurrent or malignant change of the parotid tumors.

Salvia fruticosa reduces intrinsic cellular and H_2O_2-induced DNA oxidation in HEK 293 cells;assessment using flow cytometry

Objective:To investigate the role of water-soluble extract of Salvia fruticosa(Creek sage)(S.fruticosa) leaves in reducing both intrinsic cellular and H_2O_2-induced DNA oxidation in cultured human embryonic kidney 293 cells.S.fruiicosa.native to the Eastern-Mediterranean basin,is widely used as a medicinal herb for treatment of various diseases.Methods:Dried leaves of 5.fruticosa were extracted in phosphate buffer saline and purified using both vacuum and high pressure filtrations.Each mL of the preparation contained(7.1±1.0)mg of extract.HEK-293 cells were incubated in one set with S.fruticosa extract in the presence of 0.1 mmol/L H_2O_2,and in the other set with the addition of the extract alone.The DNA oxidation was measured using fluorescence upon fluorescein isothiocyanate derivarization of 8-oxoguanine moieties.The fluorescence was measured using flow cytometry technique.Results:Cells incubated 3 h with 150μL extract and exposed to 0.1 mmol/L H_2O_2 showed lower intensity of fluorescence,and thus lower DNA oxidation.Moreover,cells incubated 3 h with 100μl.of the extract showed lower intensity of fluorescence,and thus lower intrinsic cellular DNA oxidation compared to control(without S.fruticosa).Conchisions:The results from this study suggest that the water-soluble extract of S.fruticosa leaves protects against both H_2O_2-induced and intrinsic cellular DNA oxidation in human embryonic kidney 293 cells.

A COMPARATIVE STUDY OF DNA FLOW CYTOMETRY AND MICROSPECTROPHOTOMETRY IN GASTRIC CARCINOMAS

Flow cytometric (FCM) and microspectrophotometric (MSP) measurements of cell nuclear DNA content were made in 53 fresh gastric carcinoma specimens and in 30 gastric mucosal specimens with chronic gastritis. DNA aneuploidy was found in 32/53 (60%) of gastric carcinomas, and appeared more frequently in wellor moderately differentiated tubular adenocarcinomas (90%) than in undifferentiated and mucousa cell carcinomas (23.6%) (P【0.001). No aneuploidy was found in chronic gastritis samples, but their proliferative cell fractions were higher than in normal control gastric mucosa samples (P【0.01). A comparison was made between FCM and MSP analyses of DNA content in 29 cases of gastric carcinoma, a high correlation rate (r=0.90) was found. The advantages and limitations of both methods are discussed, they may be used in combination for more precise cytochemical analysis.

FLOW CYTOMETRIC ANALYSIS OF DNA CONTENT FOR EARLY DETECTION, ESTIMATING PROGNOSIS IN BLADDER CANCER

Flow cytometry (FCM) was used to analyze the DNA content of epithelial cells in 26 cases of bladder cancer and 10 cases of nonneoplastic disease. Bladder tumor cells can be identified by aneuploidy or hyperdiploidy in histogram and/or a heterogeneity index (HI) greater than 2.30. The percentage of positive FCM in patients with bladder cancer was 84.62%. With respect to histological grading, it was 77.78% in grade 1, 90.91% in grade 2 and 100% in grade 3. While in nonneoplastic cases it was 10% Urinary exfoliative cytology was examined in all 26 patients with bladder cancer, with an accuracy of 61.54%. The DNA content of tumor cells increased with the increase of tumor grade and stage. Follow up showed that of 22 bladder cancer patients treated conservatively by transurethral resection or partial cystectomy, 8 recurred. The HI value in recurrent cases was higher than that in nonrecurrent cases. Among these 8 patients, 6 were correctly predicted by FCM. FCM appears to be an objective, sensitive and quantitative method for the diagnosis and monitoring of bladder cancer, and may be useful in estimating the prognosis of superficial bladder cancer.

FLOW CYTOM ETRIC ANALYSIS OF CELLULAR DNA CONTENT IN EPITHELIAL OVARIAN TUMOR

The DNA content of tumor all was analyzed by flow cytometry on parafflnembedded specimens in 73 patients with epithelial ovarian tumor, and its clinical significance was evaluated. One of the 5 benign (20%), 2 of the 11 borderline (18.18%), and 30 of the 57 malignant (52. 63%) tumors were aneuplold. The occurrence rate of aneuploidy In malignant tumors was higher than In benign and borderline tumors ( P < 0. 05 ). Furthermore, aneuploidy was more frequently In the advanced stages (Ⅲ -Ⅳ ) (77. 7%) than in the early stages (Ⅰ - Ⅱ ) (9. 5%) (P<0. 005). The occurrence rate of DNA aneuploidy was higher in patients associated with ascites and the residual tumor≥.2 cm. Patients with aneuploid tumors had more of ten ascites (P<0. 005) and residual tumor size≥2cm (P< 0.005). There was no apparent correlation between the DNA ptoidy and the histologic grade, histologic type of the tumors. G0/G1 cell proportion of DNA diplold tumors in advanced carcinoma (64. 6%) was less than those of early stage carcinoma (75. 9% ) (P<0. 05). The survival rate of diplold tumor patients was higher than that of aneuploid tumor patients in the different time after operation, and the median survival time was 30. 2 months and 10. 3 months, respectively. Multivariate analysis revealed that cellular DNA ploidy was the most Important predictive factor (P = 0. 007) of prognosis, followed by residual tumor size (P= 0. 05). Different tumor specimen of the same patient can exhibit variation sometime (38. 9%).The results revealed that the DNA ploidy may reflect tumor biological characteristics, I. e. , Its proliferative ability. Analysis of cellular DNA content of epithelial ovarian tumors would help us to predict the prognosis of the patients better.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes （DNA probes） were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order： PM18S02 PM28S02 〉 PM28S01 〉PM18S01, and that of the probes specific to T. pulcheUa was TP18S02 TP28S01 〉 TP28S02 〉TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02, TP18S02 and TP28S01, and the protocol, were also useful for the detection of Mgae in natural samples.

Flow Cytometric Analysis of DNA in Bone Tumors Using NucIear Suspension

The types of DNA content ean be divided into four groups:(1)diploids andnera-diploids;(2)triploids;(3)tetraploidy and hyperploidy aneuploids;(4)biclonal DHAcontent.Recent studies show that measuring DNA content by flow cytometry(FCM) can beapplied to most primary bone lumors in pointing out clinicatly relevant informattons.Ii thisstudy,cellular DHA content of 33 primary bone tvenors was analysed by FCM Isolated ratcle-ar suspensions were prepared by a simple,rapid and effective method using 10％ formatin-fctedand paraffin-embeuded bone tumor specimens.The results showed that 10 benign(inchtding 5Grade I giant cell tumors of bone) and 5 histologically questionable tumors had nornufl DNAcontent (diploids or near-diploids)and the other 7 questionable and of the 11 malignanttumors had abnormul DNA content(aneuploids).The cell cycle distribution analysis showedthat the aneuploidy tumors had higher proponion of S-phase and G2+M-phase cells than the nor-mal ploidy tumors,indicating there were differences in proliferative activity.The method alsoshowed that beniga and low-malignant primary bone tumors were diploids or near-diptoids,andhigh-malignant cnes were aneuploids Compared with typically pathological grading,the flowDNA analysis of bone tumors can more objectively point out their biological behavior andprognosis.

Correlated Flow Cytometric Analysis of H－ras p21 and DNA Ploidy in Acute Myelogenous Leukemia

The flow cytometric immunoassay was used to study the correlation between the H-ras oncogene product p21 and the DNA ploidy in 30 de novo cases of acute myelogenous leukemia (AML). The results showed that 17 cases were negative for p21 expression and 13 positive for p21. The patients with positive p21 had higher percentage of bone marrow and peripheral blasts and lower peripheral leukocyte count. The expression of p21 had no influence on the therapeutic effect. Before treatment,DNA diploidy occurred in 18 cases including 13 p21 negative ones,and DNA aneuploidy was revealed in 12 cases including 8 p21 positive ones. Patients with positive p21 or having aneuploidy in complete remission were at risk for early relapse. Our results suggest that p21 may be involved in the process of leukemogenesis and progression in AML.

Probing the Cell Cycle with Flow Cytometry

Flow cytometry is a versatile technique to study different aspects of the cell cycle from subpopulations of cells to detailed cell kinetic information. In this paper a basic review of cell kinetic parameters is presented followed by detailed descriptions of the different flow cytometric methodologies that can be used to extract pertinent information for a particular study. The methodologies range from simple DNA profile analysis, the use of bromodeoxyuridine to cell cycle-associated proteins such as the cyclins.

Spectral Compensation for Linear-Logarithmic Flow Cytometry Acquisitions

Compensating for fluorescence overlap in multiparameter flow cytometry datasets, of which one parameter is linear distributed and at least one parameter is logarithmic distributed, leads usually to extreme high compensation values. We investigated this phenomenon with an adapted flow cytometry model, of which the two parameters can easily be converted from linear to logarithmic and vice versa. With the adapted model, spectral compensation was performed both for linear-logarithmic and linear-linear parameter distribution. The results of the flow cytometry model were validated with a real world example which was also compensated twice. The results of the two experiments show that the compensation values equal to the theoretically expected value when both parameters are linear distributed. However, the compensation value exceeds 100% when one of the two parameters is logarithmic distributed. In addition, we found that spectral compensation of differently distributed parameters leads to deformation of the compensated events. With the adapted flow cytometry model presented in this paper it is shown how to correctly compensate flow cytometry acquisitions with different distributed parameters.

药用植物虎耳草的DNA条形码鉴定及基因组大小评估

目的:通过对虎耳草(Saxifraga stolonifera)进行物种鉴定和基因组大小测定,以更全面地开发其植物资源的遗传信息。方法:通过对虎耳草DNA条形码引物的筛选,发现ITS2+matK条形码可确认待测的植物样本为虎耳草。进一步利用虎耳草及其近似物种的ITS2和matK条形码序列构建系统发育树。进化分析结果表明虎耳草与其同属物种的亲缘关系较远。结果:流式细胞术估测虎耳草基因组大小约为949.4 Mb。结论:本研究基于条形码序列对十堰武当山本地的虎耳草物种进行了鉴定,为虎耳草的基因组提供了特征信息,为后续虎耳草全基因组测序研究的物种选择和测序策略规划提供了参考。

Flow cytometric investigation on degradation of macro-DNA by common laboratory manipulations

The degree and characteristics of physical degradation of macro-DNA molecules by common laboratory manipulations are reported. With linearized lambda-phage viral DNA as the model DNA, fragmentation of macro-DNA by various indispensable laboratory manipulations were investigated using a high sensitivity flow cytometric setup. Investigated manipulations included pipetting, vortexing, rocking, freeze-thawing, ultrasonication and ultrafiltration. “Exhaustive counting” of the intact lambda DNA molecules following such manipulations enabled a quantitative assessment of the resulting fragmentation, which also revealed the type of degradation reflected in the fragmentation patterns. The use of high sensitivity flow cytometry was especially suited to investigate the degradation of dilute DNA solutions that may not be suitable for analysis using traditional methods. Notable findings of this study included: the boarderline-size of DNA chains in terms of susceptibility to shear stresses by such manipulations;discernable instability of nicked DNAs;shattering-fragmentation of DNAs by freeze-thawing or ultrasonication;effectiveness of some protection media;marked “self-protection effect” of concentrated DNA solutions. These findings support and refine our traditional knowledge on how to maintain the physical integrity of macro-DNA molecules against inevitable laboratory manipulations.

药用植物冷水花DNA条形码分析和基因组大小测定

目的:鉴定采集到的冷水花属植物样本并测定其基因组大小。方法:用聚合酶链式反应扩增样品的ITS2、rbcL和psbA-trnH序列并测序,通过比对Nr核酸数据库确定物种;以玉米Mo17为对照,用流式细胞术测定其基因组大小。结果:本研究采集到的冷水花植物与冷水花(Pilea notata)ITS2序列一致性为100%,其基因组大小约为498.4 Mb。结论:ITS2序列可以较好地区分冷水花属植物,采用流式细胞术可以简便地估算待测植物样品的基因组大小。

基于流式细胞术的蜘蛛抱蛋属植物倍性检测和核DNA含量测定体系的建立

为建立适用于蜘蛛抱蛋属植物的倍性检测及其核DNA含量的流式细胞术方法,以蜘蛛抱蛋属植物成熟叶片为材料,比较了WPB解离液、LB01解离液、Galbraith’s解离液以及改良Galbraith’s解离液制备的细胞核悬液的效果。结果表明:改良Galbraith’s解离液能够更广泛地适用于蜘蛛抱蛋属植物成熟叶片的细胞核悬液的制备且效果较好。利用改良Galbraith’s解离液对12种13个居群的蜘蛛抱蛋属植物进行倍性检测,并通过染色体制片计数法进行验证。结果表明:12种蜘蛛抱蛋属植物的DNA含量的荧光强度峰值范围为1753078.81~5817826.99。其中,11种二倍体植物的峰值范围为1753078.81~2937690.80,四倍体辐花蜘蛛抱蛋的峰值为3892503.69,六倍体泰国蜘蛛抱蛋的峰值为5817826.99。此外,以葱为内标,对以上蜘蛛抱蛋属植物的核DNA含量进行测定和估算,首次估测了11种蜘蛛抱蛋植物的核DNA含量,蜘蛛抱蛋的DNA含量与已有报道的结果相近。其中,二倍体植物的核DNA含量为14.16~18.73 Gb;四倍体辐花蜘蛛抱蛋的核DNA含量为28.33 Gb;六倍体泰国蜘蛛抱蛋的核DNA含量为43.03 Gb。结果可为蜘蛛抱蛋属植物的遗传育种研究、全基因组研究以及喀斯特植物的起源与演化研究提供重要科学依据。

胸水DNA倍体联合细胞角蛋白-19片段检测对良恶性肺部疾病的鉴别诊断意义

目的探讨胸水DNA倍体联合细胞角蛋白-19片段(CYFRA-21-1)检测对良恶性肺部疾病的鉴别诊断意义。方法回顾性分析2021年5月1日至2023年12月1日浙江省荣军医院收治确诊为非小细胞肺癌伴胸水的患者78例作为病例组,同时纳入同期入院存在胸水的肺部良性疾病患者50例作为对照组。对所有患者的胸水及外周血标本进行DNA倍体及CYFRA-21-1阳性率的检测,并统计比较外周血及胸水标本中DNA倍体、CYFRA-21-1单一检测与联合检测的效能。结果病例组胸水、外周血的DNA倍体和CYFRA-21-1阳性率均明显高于对照组,差异均有统计学意义(均P<0.05);病例组胸水DNA倍体和CYFRA-21-1的阳性率均明显高于外周血,差异均有统计学意义(均P<0.05);胸水DNA倍体联合CYFRA-21-1鉴别良恶性肺部疾病的AUC为0.980,大于单一指标胸水DNA倍体的AUC 0.964和胸水CYFRA-21-1的AUC 0.900,差异均有统计学意义(均P<0.05);胸水DNA倍体联合CYFRA-21-1检测鉴别良恶性肺部疾病的灵敏度为1.00,特异度为0.96,准确度为0.98。结论肺癌患者胸水出现DNA异常增殖及CYFRA-21-1阳性率增高且远早于外周血标本,胸水DNA倍体及胸水CYFRA-21-1联合检测有助于提高良恶性肺部疾病的鉴别诊断效能,值得予以重视应用。

流式细胞仪检测高等植物细胞核DNA含量的方法

相对于动物和微生物而言,流式细胞术在植物科学上的应用会因植物组织与细胞(如细胞壁、中央液泡、特殊细胞器等)的特殊结构以及次生代谢产物等特殊成分,造成样品在前期处理、染色及测试等方面的困难,甚至导致检测失败或结果不准确。笔者在长期运用流式细胞仪测试工作中,积累了大量的植物样本检测经验,并参考国内外相关文献,总结出从植物取材、样品制备到植物细胞核DNA流式检测的方法和技巧,可为植物科学研究者及从事流式细胞检测的技术人员提供实验参考。

三个鲫品系DNA含量的比较研究

采用流式细胞术 (FCM)对红鲫、彭泽鲫、异育银鲫进行红血球DNA含量的检测分析比较 ,以鉴定它们的倍性。结果显示 ,红鲫红血球的DNA含量是 3 0pg ,彭泽鲫是 4 7pg ,异育银鲫是 4 8pg。显而易见 ,彭泽鲫的DNA含量是二倍体红鲫的 1 57倍 ,异育银鲫的DNA含量是红鲫的 1 6倍。采用肾细胞直接制作染色体的方法进行红鲫、彭泽鲫、异育银鲫的染色体倍性鉴定 ,结果红鲫的染色体数目是 10 0 ,为二倍体 (2n =10 0 ) ,彭泽鲫的染色体数目是 162 ,为三倍体 (3n =162 ) ,异育银鲫的染色体数目是 156— 162 ,为三倍体 (3n =156— 162 )。研究证明

十三种淡水养殖鱼类的DNA含量

通过流式细胞仪测定了青鱼等十三种我国主要淡水养殖鱼类外周血红细胞的ＤＮＡ含量。其中青鱼、草鱼、鲢和鳙的ＤＮＡ含量比较接近，其２Ｃ值分别为２．２０、２．１８、２．１８、２．１５ｐｇ，四种鱼相比较，差异不显著。兴国红鲤、镜鲤、荷包红鲤的ＤＮＡ含量也比较接近，其２Ｃ值分别为３．８０、３．７３、３．７６ｐｇ。相比较差异也不显著。银鲫的ＤＮＡ含量为６．０９ｐｇ／Ｎ，与鲤比接近３：２。尼罗罗非鱼和奥利亚罗非鱼的ＤＮＡ含量几乎相等，分别为２．２７、２．２０ｐｇ／Ｎ。虹鳟、鲟和团头鲂的ＤＮＡ含量分别为３．４７、１１．７３、２．６６Ｐｇ／Ｎ。由ＤＮＡ含量可见这些鱼类的血缘关系。最后讨论了测定鱼类体细胞ＤＮＡ含量的方法等问题。