[Clinical value of classified detection of circulating tumor cells in peripheral blood of NSCLC patients].

OBJECTIVE: To investigate the clinical value of detection of circulating tumor cells (CTCs) classified by epithelial cell adhesion molecule (EpCAM) in peripheral blood of patients with non-small cell lung cancer (NSCLC). METHODS: Peripheral blood samples (7.5 ml each time) were collected from 47 NSCLC patients. Among them, blood samples were collected at the end of each therapy-cycle in three patients for longitudinal monitoring of CTCs. CTCs were enriched by the depletion of leucocytes using a magnetic bead separation technique, stained with EpCAM, cytokeratin 7/8 and their isotypic control antibodies, respectively, and then identified and counted by multi-parameter flow cytometry. RESULTS: In the blood samples from 47 patients, EpCAM-positive CTCs were detected in 64.3%(9/14), 40.0%(4/10) and 43.5%(10/23) of patients in stages Ⅰ-Ⅱ, Ⅲ and Ⅳ, respectively. EpCAM-negative CTCs were detected in 78.6%(11/14), 90.0%(9/10) and 91.3%(21/23) of patients in stage Ⅰ-Ⅱ, Ⅲ, and Ⅳ, respectively. The total detection rates of EpCAM-positive and EpCAM-negative CTCs were 48.9%(23/47) and 87.2%(41/47), respectively, showing a statistically significant difference between them (P<0.001). According to the stage of the cancer, there was a significant difference between the detection rates of the two types of CTCs in patients of stage Ⅳ(P=0.001), but not in stage Ⅰ-Ⅱ and Ⅲ (P>0.05). The number of EpCAM-negative CTCs was significantly higher than that of EpCAM-positive CTCs in all stages (P<0.05). The frequency of patients with the percentage of EpCAM-negative CTCs >90% was significantly higher in stage Ⅳ patients than that in stage Ⅰ-Ⅱ cases (P=0.030), while the frequency of patients with the percentage of EpCAM-negative CTCs between 50%~90% was significantly lower in the stage Ⅳ than that in the stage Ⅰ-Ⅱ patients (P=0.001). The treatment of most patients with EpCAM-negative CTCs >50% showed to be ineffective (P=0.033). CONCLUSION: Detection of CTCs classified by EpCAM in peripheral blood is helpful in evaluating the distant metastasis and treatment effectiveness of NSCLC patients.

Two EST-derived marker systems for cultivar identification in tree peony.

Tree peony (Paeonia suffruticosa Andrews), a woody deciduous shrub, belongs to the section Moutan DC. in the genus of Paeonia of the Paeoniaceae family. To increase the efficiency of breeding, two EST-derived marker systems were developed based on a tree peony expressed sequence tag (EST) database. Using target region amplification polymorphism (TRAP), 19 of 39 primer pairs showed good amplification for 56 accessions with amplicons ranging from 120 to 3,000 bp long, among which 99.3% were polymorphic. In contrast, 7 of 21 primer pairs demonstrated adequate amplification with clear bands for simple sequence repeats (SSRs) developed from ESTs, and a total of 33 alleles were found in 56 accessions. The similarity matrices generated by TRAP and EST-SSR markers were compared, and the Mantel test (r = 0.57778, P = 0.0020) showed a moderate correlation between the two types of molecular markers. TRAP markers were suitable for DNA fingerprinting and EST-SSR markers were more appropriate for discriminating synonyms (the same cultivars with different names due to limited information exchanged among different geographic areas). The two sets of EST-derived markers will be used further for genetic linkage map construction and quantitative trait locus detection in tree peony.

[Effects of allogeneic bone marrow mesenchymal stem cells on polarization of peritoneal macrophages in rats with sepsis].

Objective: To explore the effects of allogeneic bone marrow mesenchymal stem cells (BMSCs) on polarization of peritoneal macrophages isolated from rats with sepsis induced by endotoxin/lipopolysaccharide (LPS). Methods: (1) BMSCs were isolated, cultured and purified from 5 SD rats with whole bone marrow adherent method. The third passage of cells were collected for morphologic observation, detection of expressions of stem cell surface markers CD29, CD44, CD45, and CD90 with flow cytometer, and identification of osteogenic and adipogenic differentiation. (2) Another 45 SD rats were divided into sham injury group (SI, n=5), LPS control group (LC, n=20), and BMSCs-treated group (BT, n=20) according to the random number table. Rats in groups LC and BT were injected with LPS (5 mg/kg) via tail vein to induce sepsis; rats in group SI were injected with the same amount of normal saline to simulate the damage. At post injury hour (PIH) 1, rats in group BT were given 1 mL BMSCs (2×10(6)/mL) via tail vein injection; rats in another two groups were injected with equal volume of phosphate buffer saline. Five rats in group SI at PIH 24 and in groups LC and BT at PIH 6, 12, 24, and 48 were sacrificed to harvest lung tissue for pathological observation with HE staining. In addition, rats in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48 were simultaneously performed with intraperitoneal injection of low-glucose DMEM. Then peritoneal fluid was harvested to culture peritoneal macrophages. Flow cytometer was used to assess the positive expression of cell makers of macrophages including CD68 (making gate), CD11c, and CD206 in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48. Data were processed with one-way analysis of variance and LSD test. Results: (1) The third passage of cells showed uniform fiber-like shape similar to fibroblasts. These cells showed positive expressions of CD29, CD44, CD90 and weak positive expression of CD45. They were able to differentiate into osteoblasts and adipocytes. These cells were identified as BMSCs. (2) At PIH 24, the structure of pulmonary alveoli of rats in group SI was clear and complete with no congestion or inflammatory cell infiltration. At PIH 6, the structure of pulmonary alveoli of rats in groups LC and BT was clear with a small amount of inflammatory cell infiltration, slight congestion and pulmonary interstitial thickening. At PIH 12, the inflammatory responses in lung tissue of rats in group LC were more severe than those in group BT with a large amount of inflammatory cell infiltration, serious congestion, and obvious pulmonary interstitial thickening. The pathological results of rats in group BT at PIH 12 was consistent with the results at PIH 6. At PIH 24, the pathological results of rats in groups LC and BT were similar to the results at PIH 12. At PIH 48, the structure of pulmonary alveoli tissue of rats in group LC was still severely disrupted, with a large number of inflammatory cell infiltration and congestion in lung tissue, but pulmonary interstitial thickening was slightly alleviated than before. The condition of rats in group BT nearly recovered to that in group SI. (3) At PIH 24, the positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(83±10)%] was close to that in group BT [(87±7)%, P>0.05], and they were both significantly higher than the rate in group SI [(55±12)%, with P values below 0.01]. The positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(59±11)%] at PIH 48 was close to that in group SI at PIH 24 (P>0.05), and they were both significantly higher than the rate in group BT [(20±11)%] at PIH 48 (with P values below 0.01). At PIH 24, the positive expression percentages of CD206 in peritoneal macrophages of rats were similar among the three groups (with P values above 0.05). The positive expression percentage of CD206 in peritoneal macrophages of rats in group SI at PIH 24 was close to that in group BT at PIH 48 (P>0.05), and they were both significantly lower than the percentage in group LC at PIH 48 (with P values below 0.01). Conclusions: BMSCs can reduce the pathological inflammatory responses in the lung of rats with sepsis and inhibit peritoneal macrophages from polarizing into M1 phenotype, whereas they can not promote macrophages to polarize into M2 phenotype.

[Chemical components of volatile oil from Echinops grijisii Hance].

The volatile oil obtained from Echinops grijisii roots was analysed by GC-MS method. Twenty four constituents have been identified from the oil, among which cis-beta-farnesene and 5-(3-buten-1-ynyl)bithiophene are the main ones.

Melatonin prevents pigment gallstone formation induced by bile duct ligation in guinea pigs.

Free radical-mediated oxidative stress has been implicated in the genesis of gallstone in vitro. This study was designed to examine the oxidative stress changes during pigment gallstone formation and to investigate whether melatonin (MLT) could act as a chemopreventive agent for cholelithiasis in a guinea pig model. The common bile duct of guinea pigs was ligated with or without MLT pretreatment. Animals were studied on day 7, 9, 12, and 14 after surgery. Stone and/or sludge developed in ligated guinea pigs without MLT. Fourier transform infrared spectra of the sludge showed the presence of calcium bilirubinate, whose peak height per milligram of sludge gradually increased with time after ligation. Total antioxidant activity (TAA) in bile of guinea pigs at day 14 after ligation reduced to one third of the level in sham-operated controls (P <.001). In addition, the bile of ligated guinea pigs had increased pH (P <.001), bile salts (P <.01), and malondialdehyde (MDA) (P <.05), compared to sham controls. Pretreatment of guinea pigs with MLT at a dose of 1,000 microg/kg significantly decreased the incidence of pigment gallstone formation at day 14 after ligation, as compared to no pretreatment (0/7 vs. 8/10). MLT also reverted the ligation-induced changes in biliary bile salts, pH, MDA, and TAA to control levels. These in vivo findings support a causative role of oxidative stress in the bile duct ligation-induced pigment gallstone formation. Antioxidants may prove useful in preventing pigment gallstone formation in humans.