Isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma

AIM： To explore the method of isolation and biological analysis of tumor stem cells from pancreatic adenocarcinoma cell line PANC-1. METHODS： The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 （1：1 volume） （DMEM-F12） supplemented with 20% fetal bovine serum （FBS）. Subpopulation cells with properties of tumor stem cells were isolated from pancreatic adenocarcinoma cell line PANC-1 according to the cell surface markers CD44 and CD24 by flow cytometry. The proliferative capability of these cells in vitro were estimated by 3-[4,5-dimehyl-2-thiazolyl]-2, 5-diphenyl- 2H-tetrazolium bromide （MTT） method. And the tumor growth of different subpopulation cells which were injected into the hypodermisof right and left armpit of nude mice was studied, and expression of CD44 and CD24 of the CD44^＋CD24^＋ cell-formed nodules and PANC-1 cells were detected by avidin-biotin-peroxidase complex （ABC） immunohistochemical staining. RESULTS： The 5.1%-17.5% of sorted PANC-1 cells expressed the cell surface marker CD44, 57.8% -70.1% expressed CD24, only 2.1%-3.5% of cells were CD44^＋ CD24^＋. Compared with CD44-CD24- cells, CD44^＋CD24^＋ cells had a lower growth rate in vitro. Implantation of 104 CD44 CD24 cells in nude mice showed no evidenttumor growth at wk 12. In contrast, large tumors were found in nude mice implanted with 103 CD44^＋CD24^＋ cells at wk 4 （2/8）, a 20-fold increase in tumorigenic potential （P 〈 0.05 or P 〈 0.01）. There was no obvious histological difference between the cells of the CD44^＋CD24^＋ cell-formed nodules and PANC-1 cells. CONCLUSION： CD44 and CD24 may be used as the cell surface markers for isolation of pancreatic cancer stem cells from pancreatic adenocarcinoma cell line PANC-1. Subpopulation cells CD44^＋CD24^＋ have properties of tumor stem cells. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, it may be a very promising target for new dr     

氯法齐明抗结核分枝杆菌的体内外活性研究

目的 评价氯法齐明(clofazimine,CLF)在体外及体内抗结核分枝杆菌的活性,为临床应用提供依据.方法 应用微孔板指示剂法,测定氯法齐明对结核分枝杆菌标准株H37Rv及临床分离耐多药结核分枝杆菌(30株)的MIC;建立小鼠静脉感染H37Rv的结核病模型(105 CFU/只),根据氯法齐明的不同剂量,分为3个治疗组:20 mg/kg,每周给药5次(CLF-1组);10 mg/kg,每周给约5次(CLF-2组);20 mg/kg,每周给药2次(CLF-3组);治疗30 d后进行肺、脾组织活菌计数,应用单冈素方差分析,比较氯法齐明在小鼠体内的抗结核分枝杆菌活性.结果 氯法齐明对结核分枝杆菌H37Rv的MIC为0.12～0.24 μg/ml;对30株耐多药结核分枝杆菌临床分离株的MIC为0.12～1.92 μg/ml.在小鼠结核病治疗模型中,3个治疗组的小鼠肺活菌计数分别较空白对照组降低2.92、1.78和1.39 lg CFU,均具有统计学意义(F=74.09,P＜0.01).结论 氯法齐明具有较好的体外及体内抗结核分枝杆菌活性,值得进一步研究.     

嗜酸细胞性胃肠炎临床分析

嗜酸细胞性胃肠炎（EG）是一种少见的良性消化道疾病，临床表现无特异性，误诊率较高。目的：通过对EG的一般情况、发病诱因、临床表现、辅助检查、诊断分型、误诊情况以及治疗和预后进行分析，以提高对该病的认识，避免误诊误治，减少患者痛苦。方法：对中国人民解放军总医院消化疾病中心的4例EG以及以“嗜酸细胞。胃肠炎”为关键词在中国医院知识仓库医学专题全文数据库（CHKD）中检索得到的1997～2007年发表的文献中169例EG的临床特点进行分析。结果：本组资料显示，44．5％的EG与过敏有关，常见临床表现为腹痛（94，2％）、腹泻（60．7％）、恶心（56．1％）、呕吐（51．5％）、腹胀（38-2％）、腹水（28．3％）。嗜酸性粒细胞（EOS）升高在外周血、骨髓和腹水中的阳性率分别为74．0％、98，2％和90．3％。胃镜和结肠镜黏膜活检发现EOS浸润胃黏膜和肠黏膜分别占88-2％和64．6％。诊断以黏膜型EG最为多见（65．3％），误诊率为40．5％。77．5％的EG患者接受糖皮质激素治疗，疗效良好。结论：EG是一种易误诊的少见病。临床表现无特异性，确诊应从提高对该病的认识、适时行血常规、骨髓和腹水EOS检查、胃肠道黏膜活检病理检查等方面人手。糖皮质激素治疗EG有效，预后良好。     

胃黏膜脱垂与慢性胃炎关系探讨

目的研究胃黏膜脱垂(prolapseofgastricmucosa ,GMP)与慢性胃炎的关系。方法选取二组接受胃镜检查的患者①慢性胃炎组,均符合1996年悉尼慢性胃炎诊断标准,并除外胃、十二指肠溃疡性疾病及十二指肠球部变形者。共10 3例,男5 7例,女46例。年龄2 2～74岁,平均年龄48 9岁。②对照组同期接受胃镜检查的患者,未发现胃、十二指肠存在活动性炎症、溃疡等病变表现,亦为10 3名,男60名,女43名。年龄2 0～76岁,平均年龄47 3岁。结果①共69例( 3 3 .5 % )存在GMP ,其中慢性胃炎患者及对照组中分别为43例( 4 1.7% )及2 6例( 2 5 .2 % ) ,慢性胃炎组中GMP检出率显著高于对照组(P <0 .0 5 )。②GMP在各年龄组,男女间发病率无显著性差异(P >0 .0 5 )。③GMP患者中Helicobecterpylori检测阳性率( 10 7% )显著低于GMP阴性患者H .pylori检出率( 2 6.2 % ) (P <0 .0 5 )。④43例伴GMP的慢性胃炎中胃窦条状红斑( 3 0 .2 % )、食管炎( 16.3 % )均高于60例不伴GMP中相应比例( 10 .0 % ,P <0 .0 1;3 .3 % ,P <0 .1)。结论GMP与慢性胃炎相关,胃窦条状红斑为特征性表现,GMP患者易合并食管炎;GMP发生与性别年龄无关;GMP患者H .pylori感染率较低。     

Comparison of the Masaoka-Koga staging and the International Association for the Study of Lung Cancer/the International Thymic Malignancies Interest Group proposal for the TNM staging systems based on the Chinese Alliance for Research in Thymomas retrospective database

Background

To compare the predictive effect of the Masaoka-Koga staging system and the International Association for the Study of Lung Cancer (IASLC)/the International Thymic Malignancies Interest Group (ITMIG) proposal for the new TNM staging on prognosis of thymic malignancies using the Chinese Alliance for Research in Thymomas (ChART) retrospective database.

Methods

From 1992 to 2012, 2,370 patients in ChART database were retrospectively reviewed. Of these, 1,198 patients with complete information on TNM stage, Masaoka-Koga stage, and survival were used for analysis. Cumulative incidence of recurrence (CIR) was assessed in R0 patients. Overall survival (OS) was evaluated both in an R0 resected cohort, as well as in all patients (any R status). CIR and OS were first analyzed according to the Masaoka-Koga staging system. Then, they were compared using the new TNM staging proposal.

Results

Based on Masaoka-Koga staging system, significant difference was detected in CIR among all stages. However, no survival difference was revealed between stage I and II, or between stage II and III. Stage IV carried the highest risk of recurrence and worst survival. According to the new TNM staging proposal, CIR in T1a was significantly lower comparing to all other T categories (P<0.05) and there is a significant difference in OS between T1a and T1b (P=0.004). T4 had the worst OS comparing to all other T categories. CIR and OS were significantly worse in N (+) than in N0 patients. Significant difference in CIR and OS was detected between M0 and M1b, but not between M0 and M1a. OS was almost always statistically different when comparison was made between stages I–IIIa and stages IIIb–IVb. However, no statistical difference could be detected among stages IIIb to IVb.

Conclusions

Compared with Masaoka-Koga staging, the IASLC/ITMIG TNM staging proposal not only describes the extent of tumor invasion but also provides information on lymphatic involvement and tumor dissemination. Further study using prospectively recorded information on the proposed TNM categories would be helpful to better grouping thymic tumors for predicting prognosis and guiding clinical management.     

Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM

The radial spoke (RS) heads of motile cilia and flagella contact projections of the central pair (CP) apparatus to coordinate motility, but the morphology is distinct for protozoa and metazoa. Here we show the murine RS head is compositionally distinct from that of Chlamydomonas. Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a and Rsph10b, whose orthologs exist in the protozoan RS head. We resolve its cryo-electron microscopy (cryo-EM) structure at 3.2-Å resolution. Our atomic model further reveals a twofold symmetric brake pad-shaped structure, in which Rsph4a and Rsph9 form a compact body extended laterally with two long arms of twisted Rsph1 β-sheets and potentially connected dorsally via Rsph3b to the RS stalk. Furthermore, our modeling suggests that the core complex contacts the periodic CP projections either rigidly through its tooth-shaped Rsph4a regions or elastically through both arms for optimized RS–CP interactions and mechanosignal transduction.

The majority of motile cilia and flagella are composed of nine dynein arm-containing peripheral doublet microtubules (DMTs) surrounding a central pair (CP) of MTs (the “9+2” axoneme). The radial spoke (RS) is a T-shaped protein complex with an orthogonal head pointing toward the CP and a stalk anchored on each A-tubule of the DMTs (1–5). It acts as the mechanochemical transducer between the CP and axonemal dynein arms to regulate flagellar/ciliary motility (6–11). The flagella of Chlamydomonas reinhardtii, a widely used model organism, contain two full-size RSs (RS1 and RS2) in each 96-nm repeat unit of the axoneme. In contrast, motile cilia/flagella of Tetrahymena thermophila and metazoa possess triplet RSs (RS1 to RS3) (2–4, 11). The Chlamydomonas RS is composed of at least 23 subunit proteins (RSP1 to RSP23) (2, 12, 13). Seventeen of them have mammalian homologs (14). Mutations leading to the loss of the entire RS or RS head result in immotile flagella in Chlamydomonas (6–8) but in rotatory ciliary beat in mammals, causing primary ciliary dyskinesia (PCD), a genetic syndrome characterized by recurrent respiratory infections, situs inversus, infertility, and hydrocephalus (4, 15–21).The most striking morphological differences in the RS lie in the RS head, the key structural domain that mediates the mechanosignaling by directly contacting projections of the CP (9–11). The heads of RS1 and RS2 consist of two structurally identical, rotationally symmetric halves that differ largely from that of RS3 (3, 4). Furthermore, their morphologies differ dramatically between protozoa and metazoa. In Chlamydomonas and Tetrahymena, for instance, the heads of RS1 and RS2 are rich in lateral branches that also form a connection between the two heads (2, 4). In contrast, in sea urchin (Strongylocentrotus purpuratus) and human, the heads of RS1 and RS2 resemble a pair of ice skate blades with many fewer interfaces toward the CP (3, 4). Despite the importance of the RS and RS head in cilia/flagella motility, the structural details of the RS and the RS–CP interactions remain poorly understood, especially in mammals.The RS heads have probably been remodeled to comply with both structural and functional alterations of the axoneme during evolution. How the morphological changes occurred, however, remains unclear. The Chlamydomonas RS head is composed of RSP1, -4, -6, -9, and -10 and part (the C terminus) of the stalk component, RSP3. Each of the symmetrical halves of the head contains one copy of these components (2, 10, 22). All the head components have mammalian orthologs (Rsph1, -4a, -6a, -9, -10b, and -3b) (11, 14). In sharp contrast to the markedly reduced surface area of metazoan RS heads, the peptides of human RSPH4A, -6A, and -10B are longer than their Chlamydomonas orthologs by 1.5-, 1.3-, and 4-fold, respectively (11). Only RSPH1 (309 amino acids [aa]) is shorter than RSP1 (814 aa) (11). The lengths of mouse RS head proteins are also similarly changed as their human counterparts (SI Appendix, Fig. S1A). Furthermore, while murine Rsph4a is essential for the head formation of RS1 to RS3 in motile multicilia of the trachea, ependyma, and oviduct (15), Rsph6a is specifically expressed in sperm for their normal flagellar formation (23). RSP4/Rsph4a and RSP6/Rsph6a are paralogs: RSP4 and RSP6 share 48% sequence identity (24), whereas murine Rsph4a is 63% identical to Rsph6a (SI Appendix, Fig. S1B). Sea urchin and Ciona, however, have only one ortholog (11, 25). These results suggest that, unlike the protozoan RS heads, the metazoan ones may not simultaneously contain Rsph4a and Rsph6a. The general shapes of the RS structure in axonemes have been determined by conventional electron microscopy (EM) (26–28) and cryo-electron tomography (cryo-ET) (2–5). Recently, a 15-Å-resolution RS structure of Chlamydomonas was resolved by cryo-EM single-particle analysis (29). The resolutions, however, do not suffice for the delineation of the locations of individual RS subunits.In the present study, by biochemical and structural analyses, we show the murine RS head is both compositionally and morphologically distinct from that of Chlamydomonas. Our study suggests that the RS head has experienced profound remodeling to probably comply with both structural and functional alterations of the axoneme during evolution for coordinated ciliary or flagellar motility.     

Effect of b value on monitoring therapeutic response by diffusion-weighted imaging

AIM： To explore the diffusion gradient b-factor that optimizes both apparent diffusion coefficient （ADC） measurement and contrast-to-noise （CNR） for assessing tumor response to transarterial chemoembolization （TACE） in a rabbit model. METHODS： Twelve New Zealand white rabbits bearing VX2 tumors in the liver were treated with TACE. Diffusion-weighted imaging （DWI） with various b values was performed using the same protocol before and 3 d after treatment with TACE. ADC values and CNR of each tumor pre- and post-treatment with different b factors were analyzed. Correlation between ADC values and extent of necrosis in histological specimens was analyzed by a Pearson＇s correlation test.RESULTS： The quality of diffusion-weighted images diminished as the b value increased. A substantial decrease in the mean lesion-to-liver CNR was observed on both pre- and post-treatment DW images, the largest difference in CNR pre- and post-treatment was manifested at a b value of 1000 s/mm^2 （P = 0.036 ）. The effect of therapy on diffusion early after treatment was shown by a significant increase in ADCs （P = 0.007）, especially with large b factors （≥ 600 s/mm^2）. The mean percentage of necrotic cells present within the tumor was 76.3%-97.5%. A significant positive correlation was found between ADC values and the extent of necrosis with all b values except for b200, a higher relative coefficient between ADC values and percentage of necrosis was found on DWI with bl000 and b2000 （P = 0.002 and 0.006, respectively）. CONCLUSION： An increasing b value of up to 600 s/mm^2 would increase ADC contrast pre- and post-treatment, but decrease image quality. Taking into account both CNR and ADC measurement, diffusion-weighted imaging obtained with a b value of 1000 s/mm^2 is recommended for monitoring early hepatic tumor response to TACE.     

L－精氨酸对急慢性缺氧大鼠肺循环和脑血流的影响

研究了Ｌ－精氨酸（Ｌ－Ａｒｇ）对急慢性缺氧大鼠肺循环和脑血流的影响。一次注射Ｌ－Ａｒｇ４００ｍｇ·ｋｇ￣（－１）对急性缺氧性肺血管收缩反应（ＨＰＶ）无影响，而８００ｍｇ·ｋｇ￣（－１）有明显抑制作用；两种剂量的Ｌ－Ａｒｇ对缺氧性脑血流的变化均无显著影响。Ｌ－Ａｒｇ（３００ｍｇ·ｋｇ￣（－１）·ｄ￣（－１））能缓解慢性缺氧性肺动脉高压、肺血管阻力增高和右心肥厚，并抑制ＨＰＶ，但对脑血流无显著影响。ＮＯ合成酶抑制剂Ｌ－精氨酸甲酯可拮抗Ｌ－Ａｒｇ的作用，提示Ｌ－Ａｒｇ的作用可能与增加ＮＯ合成有关。     

缬沙坦长期治疗对原发性高血压患者左心室肥厚及心功能影响的对比研究

目的探讨血管紧张素Ⅱ受体拮抗剂--缬沙坦对原发性高血压患者左心室肥厚及心功能的影响.方法入选经超声心动图检查证实为原发性高血压的左心室肥厚患者72例,随机分配到缬沙坦组(口服80～160mg/d)或阿替洛尔组(口服25～50 mg/d)(n均=36),治疗8个月,治疗前后各检查一次超声心动图及放射性核素心室显像,对比分析组内治疗前后左心室重量指数及左心功能参数变化和两组间的差异.结果①与治疗前比较,原发性高血压患者在缬沙坦或阿替洛尔治疗8个月后,两组收缩压与舒张压明显下降(159/101 mmHg至142/89 mmHg;161/103 mmHg至145/90mmHg,1 mmHg=0.133 kPa)(P均＜0.01).②原发性高血压患者在缬沙坦治疗8个月后,左心室后壁与室间隔厚度较治疗前显著下降(P均＜0.05),左心室重量及左心室重量指数下降更显著(P均＜0.01);而在阿替洛尔治疗8个月后左心室后壁与室间隔厚度无明显变化,左心室重量及左心室重量指数下降显著(P＜0.05).③原发性高血压患者在缬沙坦治疗8个月后左心室高峰充盈率明显增加(P＜0.05),而在阿替洛尔治疗8个月后左心室高峰充盈率无明显变化,两组间比较则有明显差别(P＜0.05).结论原发性高血压患者在缬沙坦治疗8个月后可使原发性高血压患者左心室肥厚显著逆转及左心室舒张功能显著改善,缬沙坦对左心室舒张功能的作用优于阿替洛尔.