Objective:Use the droplet digital PCR (ddRCR) technology to establish, optimize and evaluate the method of EGFR-T790M mutation detection.Methods:The relevant probes and primers were designed for EGFR-T790M mutations. The ddPCR reaction system was established, the optimal annealing temperature was set and the basic performance of the method was tested. On this basis, from January 2019 to October 2019, 72 cell-free DNA (cfDNA) samples from NSCLC patients were collected from Shandong Cancer Hospital Affiliated to Shandong First Medical University, and clinically verified. The consistency of the gene mutation detections with Bole ddPCR products was analyzed using Kappa test.Results:The ddPCR reaction system was established and optimized. Linear evaluation showed the R2 value was greater than 0.99. Using ddPCR, the blank detection limit was determined to be the numbers of mutant droplets≥2, with excellent specificity. For the sensitivity analysis, the lower limit of mutation detection was determined to be at least 0.05%. In the repeatability and inter-assay precision tests, the results had a coefficient of variation( CV)<20%. The relative deviation of the results was within the range of±10% for the accuracy analysis. Using the established T790M mutation detection method, 72 samples from the NSCLC patients were tested for genetic mutation in cfDNA, and the overall agreement with the Bole ddPCR products was 91.67% (66/72, Kappa=0.749; P<0.001). Conclusion:Using ddPCR, the method of EGFR-T790M mutation detection for NSCLC was successfully established.

Objective To explore the clinical value of magnetic resonance imaging( MRI) in the diagnosis and classification of perianal abscess and anal fistula.Methods Eighty patients with suspected perianal abscess and anal fistula were selected in Yuyao People＇s Hospital from May 2016 to December 2017.They were divided into general examination group and MRI group by random number table,with 40 cases in each group. The general examination group underwent ultrasound examination,and the MRI group underwent MRI examination.The detection rate of MRI examination for various types of perianal abscess and anal fistula was analyzed based on the results of operation. Results In the general examination group, the diagnostic accuracy of anal fistula supervisor, anal fistula internal orifice,perianal abscess and anal fistula branch were 65.0% (26/40),70.0% (28/40),57.5% (23/40) and 52.5% (21/40),respectively,which in the MRI group were 92.5% (37/40),77.5% (31/40),87.5% (35/40) and 95.0% (38/40),respectively.There were statistically significant differences in diagnostic accuracy of anal fistula supervisor, perianal abscess and anal fistula branch between the two groups ( χ2 =9.054,7.116,8.865, all P <0.05). The effective rate of operation in the MRI group was 67.5% (27/40),which was significantly higher than that in the general examination group [67.5% (27/40)],the difference was statistically significant(χ2 =9.935,P<0.01). Conclusion Using MRI to diagnose perianal abscess and anal fistula can accurately judge the number,involvement range,specific location and surrounding structure of the abscess and anal fistula,and has important value for clinical treatment and prognosis.

BACKGROUND: During the occurring and developing of tumor, tumor-educated platelets mRNA profiles were altered. Since platelets are anuclear, the level of mRNAs is probably post-transcriptional regulated by the splicing maturation of pre-mRNA and alternative splicing. Apoptotic chromatin condensation inducer 1 (ACIN1) has been shown to be a component of a splicing-dependent multiprotein exon junction complex (EJC) and was involved in mRNA metabolism associated with splicing. This study analyzed the expression of ACIN1 mRNA in platelets, and explored its potential as a biomarker of lung cancer. METHODS: 156 patients with lung cancer and 58 healthy controls in Shandong Cancer Hospital were collected. We isolated platelet pellets by low-speed centrifugation and extracted total RNA. The expression of ACIN1 mRNA in platelets was detected by RT-PCR, the results were analyzed statistically. And the relationship between expression of ACIN1 mRNA and clinical factors were also analyzed. RESULTS: The expression level of ACIN1 mRNA in platelets of patients with lung cancer was significantly higher than that in platelets of healthy controls (P=0.015). The ROC curve showed that the area under the curve of ACIN1 mRNA for detecting lung cancer were 0.608. The expression of ACIN1 mRNA in platelets of lung cancer has no significant relationship with age, gender, pathological type and metastasis or not (P>0.05). CONCLUSIONS ACIN1 mRNA was highly expressed in platelets of lung cancer patients, and the detection of its expression level might have potential clinical value for the diagnosis of lung cancer.
Blood Platelets ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; blood ; genetics ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo observe the effects of heat-sensitive moxibustion on corticotropin releasing hormone (CRH), adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) in rats with irritable bowel syndrome (IBS), and to explore the possible mechanism of heat-sensitive moxibustion on IBS.

METHODSAccording to random number table, 56 SD male rats were randomly divided into a blank group (=8), a model group (=8), a moxibustion group (=32), and a mifepristone group (RU-486 group,=8). The rats in the blank group were treated with normal feeding; the rats in the model group, RU-486 group and moxibustion group were treated with chronic non-predictable stimulation for 21 days to establish IBS model. After model establishment, the rats in the moxibustion group were treated with moxibustion at "Mingmen" (GV 4) for 40 min, once a day for 14 days; the tail temperature was recorded every 5 min; according to the change of tail temperature, the rats were divided into a heat-sensitive moxibustion group and a non-heat-sensitive moxibustion group, and 8 rats were randomly selected in the two groups. The rats in the RU-486 group were treated with gastric administration of RU-486 for 14 days, while the rats in the blank group, model group and moxibustion groups were treated with identical volume of 0.9% NaCl. The rat general condition, body mass, behavioristics, intestinal propulsive rate and visceral sensitivity were observed in each group; ELISA method was used to detect serum CRH, ACTH and CORT; optical microscope was applied to observe the morphological changes of colon.

RESULTS(1) After model establishment, rats were in rest state, fatigued, with withered hair and dim ear; the stool was dry or watery; the body mass were slowly increased; the number of crossed grid and standing frequency were significantly reduced; visceral sensitivity was increased and intestinal propulsion rate was decreased; no obvious inflammatory cell infiltration was observed under microscope. (2) After intervention, compared with the blank group, the body mass and visceral sensitivity in the RU-486 group were not significantly different (both>0.05), but the intestinal propulsion rate was decreased significantly (<0.01). Compared with the blank group, the body mass of heat-sensitive moxibustion group and non-heat-sensitive moxibustion group was lower (both<0.01), but the visceral sensitivity and intestinal propulsion rate were similar (both>0.05). Compared with the model group, the body mass and visceral sensitivity were improved in the RU-486 group (<0.05,<0.01), but the intestinal propulsion rate was similar (>0.05). The body mass, visceral sensitivity and intestinal propulsion rate of the heat-sensitive moxibustion group and the non-heat-sensitive moxibustion group were superior to those of the model group (<0.05,<0.01), and the body mass and intestinal propulsion rate of heat-sensitive moxibustion group were superior to those of non-heat-sensitive moxibustion group (both<0.05). (3) After intervention, compared with the blank group, the contents of CRH, ACTH and CORT in the model group were significantly increased (<0.05,<0.01). Compared with the model group, the contents of CRH, ACTH and CORT of the heat-sensitive moxibustion group were statistically reduced (<0.05,<0.01), and the contents of CRH and ACTH in the non-heat-sensitive moxibustion group were statistically reduced (<0.05,<0.01); the content of CRH in the RU-486 group was reduced (<0.05), but the contents of ACTH and CORT were increased (<0.05,<0.01). Compared with the non-heat-sensitive moxibustion group, the heat-sensitive moxibustion group was better in the improvement of CRH (<0.05), but there was no significant difference of ACTH and CORT between the two groups (both>0.05).

CONCLUSIONHeat-sensitive moxibustion could reduce the contents of CRH, ACTH and CORT through the HPA axis, and improve the function of gastrointestinal motility to treat IBS.

Pharmaceutical preparations can directly affect the administration methods and therapeutic effects of drugs , which is a priority for the research and development of the military specialized medicament .Foreign armies started pharma-ceutical formulation research very early , and some of their research concepts and strategies are worth learning from .In this paper , dosage forms were used as the classification factor and several formulations with distinct military characteristics were described in detail .The features of military specialized medicament were analyzed from the perspective of pharmaceutics , based on which future development in the formulation of military specialized medicament was predicted .

Despite tre mendous research efforts have been devoted to the analysis of nanoparticles (NPs)biohazard,the potential mechanism for nanotoxicity has not yet been syste mati-cal y elucidated.This review intends to point out the confusions about nanotoxicity in the field and tries to look into the mecha-nism from a new perspective.Currently,there are three puzzles:① no relationship between dose and toxicity could be observed in nanotoxicity;②there is a theory for the″size effects″,however, it cannot explain some cases contrary to the doctrine;③ NPs made of different materials with various sizes could have the same toxic effects through sti mulating oxidative stress.In fact, human body is co mposed of various biological molecules,and the biological function of a living syste m is reflected by the inter-actions and conversions of those molecules.NPs,on the other hand,are the invader of human body which has no ability to transport or convert or digest the foreigner.Thus,NPs could cause celldamage due to the physical blockage of micro-circula-tion,celldestruction due to membrane rando m insertion,and celldysfunction due to physical contacting with big biological mole-cules.The physical damages caused by various NPs could be divided into three categories:adhesion lesion,card inlay and puncture.Above al ,by analyzing wide spectrum of NPs varying in co mposition,shape and size,the author draws a conclusion that physical damage is the origin of nanotoxicity.

Objective To compare the pressure-controlled ventilation (PCV) and volume-controlled ventilation (VCV) in the patients undergoing spinal surgery in prone position supported by a Wilson frame.Methods Forty patients,of ASA physical status Ⅰ or Ⅱ,aged 30-64 yr,with body mass index ＜ 30 kg/m2,scheduled for elective spinal surgery in prone position supported by a Wilson frame under general anesthesia,were randomly allocated to receive mechanical ventilation using either VCV (n =20) or PCV (n =20) mode.Endotracheal intubation and mechanical ventilation were performed after induction of anesthesia.The tidal volume (VT) was set at 10 ml/kg according to the ideal body weight in group VCV.The maximal inspiratory pressure of the anesthesia machine was adjusted to maintain the VT at 10 ml/kg in group P.Both ventilation modes were required to maintain PET CO2 within the normal range.VT,respiratory rate,minute ventilation (MV),dynamic lung compliance (Cdyn),peak and mean airway pressure (Ppeak,Pmean),mean arterial pressure (MAP) and HR were recorded at 10 min after the patients were turned to supine position and at 30 min after the patients were turned to prone position after intubation.Arterial blood samples were collected for blood gas analysis,and oxygenation index(OI) and physiologic dead space fraction (VD/VT) were calculated.Results Compared with those at 10 min after turning to supine position,Ppeak was significantly increased and Cdyn,VT and MV were decreased at 30 min after turning to prone position in both groups.Compared with group VCV,Ppeak was significantly decreased,respiratory rate and Cdyn were increased,and no significant change was found in VT,MV,OI,VD/VT,Pmean,MAP and HR in PCV group.Conclusion Compared with VCV,PCV can improve the ventilatory efficacy and reduce the influence of prone position on respiratory dynamics in the patients undergoing spinal surgery in prone position supported by a Wilson frame.

Objective To investigate the pathologic mechanism of radiofrequency ablation ( RFA ) combined with intravenous infusion of thermosensitive liposome encapsulated vinorelbine (TL-Vin) in treating liver tumors, and to analyze the effect of combination therapy on the long-term survival rate. Methods H22 liver adenocarcinoma tissue was subcutaneously implanted into ICR mice to establish the animal models. At the first experimental period, 40 mice were randomly and equally divided into 5 groups to receive different therapeutic scheme (using different TL-Vin concentrations). Twenty-four hours after the treatment the tumor specimens were collected, the necrotic areas were measured separately, and the optimal TL-Vin concentration was determined. At the second experimental period, 13 mice were randomly selected to receive treatment. Half an hour after the treatment the tumor tissues were collected and the TL-Vin concentration within the tumor was determined. At the third experimental period, 32 mice were randomly and equally divided into 4 groups, and 90 days after treatment the tumor growth curve was drawn. The survival rate was compared between each other of the groups. Results Compared with pure RFA group, TL-Vin + RFA significantly increased tumor coagulation extent (P<0.01). But free-VIN+RFA had similar tumor necrotic extent as that produced by RFA alone (P>0.05). Tumor coagulation area in TL-Vin + RFA group was bigger than that in free-VIN + RFA group at the concentration of 10 mg/kg [(341.8 ± 65.4)mm2 vs (225.3 ± 25.4)mm2, P < 0.01]. In TL-Vin group the coagulation margin was clear. The mean intratumoral Vinorelbine accumulation in TL-Vin + RFA group was 10 folds of that in free-Vin group [(1 156.5 ± 158.3)ng/ml vs (194.5 ± 52.3)ng/ml, P = 0.005]. TL-Vin +RFA had better survival result than that of RFA alone (37.6 ± 20.1 days vs. 23.4 ± 5.0 days, P=0.015), as well as than that of free-Vin + RFA [(37.6 ± 20.1)days vs (23.3 ± 1.2)days, P = 0.016]. Conclusion Thermosensitive liposomal chemotherapies (Vinorelbine) can be selectively delivered at the edge of RFA coagulation area and thus effectively increase RFA-induced tumor coagulation and prolong the end-point survival in experimental mice.

An on-line solid phase extraction ( SPE ) coupled with HPLC-MS/MS method was developed to determine S-ammuxetine and R-ammuxetine in rat plasma. The sample preparation consisted of the following steps:A protein precipitation extraction used methanol and acetonitrile ( 50:50 , V/V ); an on-line SPE treatment to remove most matrixes in plasma;an enrichment and separation step used a C18 analytical column. S-and R-ammuxetine were determined by tandem mass spectrometry. The SPE column was a Retain PEP Javelin (10 mm × 2. 1 mm × 5 μm), while the chromatographic separation was achieved using a ZORBAX SB-C18 (50 mm × 2. 1 mm × 3. 5 μm) analytical column with an isocratic mobile phase composed of acetonitrile-water-formic acid (40:60:0. 1, V/V/V, 0. 3 mL/min). The selected reaction monitoring mode of the positive ion was performed and the precursor to the product ion transitions of m/z 292 . 1/154 . 0 and m/z 260. 4/116. 2 were used to measure S-ammuxetine, R-ammuxetine and internal standard (propranolol). The method was linear over a concentration range from 0 . 2 to 1000 μg/L with the correlation coefficients of 0 . 9903 and 0 . 9951 . The average intra-day precision values were 1 . 2% -12 . 0% for S-ammuxetine and 0. 4%-11. 2% for R-ammuxetine, respectively. The average recoveries were 94. 2%-101. 6% for S-ammuxetine and 94. 3% -109. 4% for R-ammuxetine. Compared to the literature, the sensitivity of this method increased dramatically. The present method has been successfully applied to the preclinical rat research of ammuxetine isomers following intragastric administration.

Liposomes can be cleared by the reticuloendothelial system (RES) when it is in the blood circulation in the body. And they can accumulate in the organs rich in RES in the body by passive targeting. Targeting of the liposomes is an important factor for its use as a drug carrier, and particle size as well as surface charge are important for its in vivo targeting. In this paper, studies on the influences of particle size and surface charge of the liposomes on cell binding and phagocytosis mechanism were reviewed. A comprehensive review on passive targeting effect of the particle size and surface charge of liposomes on blood, liver, spleen as well as tumor tissue was made. At last, an outlook for future research directions was made.