BACKGROUND:Very smal embryonic-like stem cells are a kind of non-hemopoietic stem cells, which have similar biological characteristics to embryonic stem cells. But the method of its culture and in vitro proliferation is rarely reported. Studies have speculated that human embryonic fibroblasts can provide a good microenvironment for in vitro culture and proliferation of very smal embryonic-like stem cells.
OBJECTIVE:To isolate and cultivate human embryonic fibroblasts derived from human embryonic trunks and to establish a feeder layer culture system of human embryonic fibroblasts for culturing very smal embryonic-like stem cells derived from human bone marrow.
METHODS:The human embryonic fibroblasts were isolated from the subcutaneous connective tissue of human embryos at pregnant 5-9 weeks using trypsin digestion method. Different concentrations of mitomycin C were used to pretreat feeder layers, which were used for cultivating very smal embryonic-like stem cells derived from human bone marrow. The effects of human embryonic fibroblasts and feeder layers were assessed by cel morphology and growth curves.
RESULTS AND CONCLUSION:The human embryonic fibroblasts were successful y isolated and cultivated from human embryos, and they could be passaged beyond the 24th generation. The biologic characteristics of the cells had no changes after passage and cryopreservation. The optimal concentration of mytomcin C to inhibit proliferation of human embryonic fibroblasts was l2 mg/L for 3 hours. The human embryonic fibroblasts derived from human embryos are successful y isolated and cultivated and to produce feeder layers for very smal embryonic-like stem cells derived from human bone marrow.

OBJECTIVE To obtain the statistics of the resistance of Staphylococcus spp to 12 antibacterial agents. METHODS The clinical non-repeated isolates of Staphylococcus aureus and coagulase negative Staphylococcus(CNS) were collected in September 2004 in 13 hospitals of Anhui Province.According to National Committee for Clinical Laboratory Standards(NCCLS) of America(2004),agar dilution method was performed to determine the MICs of 12 antimicrobial agents against the isolates. RESULTS All S.aureus and 92.9% of CNS were resistant to penicillin respectively.Multi-resistance to antibiotics such as macrolides,and fluoroquinolones were found in these strains,and all the isolates of Staphylococcus spp were susceptible to vancomycin. CONCLUSIONS Staphylococcus spp exhibit some degrees of resistance to antimicrobial agents in Anhui Province.Therefore,the drug resistance of surveillance must be imposed,and we must emphasize on reasonable option of antimicrobial agents in clinical therapy in our area.

By the survey of topic selection of medical master's dissertation for candidates with the same educational level,the writers think that the decided topics should be in close relation to their respective subjects and the operational and technical conditions of their institutes and the training institutes should be taken into full consideration.The decided topics should enhance theoretical and practical values and will be achieved in the required time as well.

Objective To dynamic monitor and analyze the characteristic of polysomnography (PSG)and melatonin levels of delirium patients in intensive care unit (ICU). Methods A prospective observational study was performed from December 2013 to April 2014. The patients admitted to ICU of Affiliated Hospital of Binzhou Medical College for more than 72 hours were evaluated with confusion assessment method for the ICU (CAM-ICU),and were divided into delirium group and non-delirium group. Sleep patterns of all the patients underwent continuous PSG for up to 24 hours were evaluated. Melatonin levels were determined every 4 hours with enzyme linked immunosorbent assay (ELISA)duration sleep monitoring. Results Eighteen patients were enrolled,and 9 were delirium patients. All the patients had sleep disorders:a decrease in rapid eye movement (REM)sleep 〔(5.91±5.26)%〕,an increase in the sleep fragmentations 〔arousal index was (15.40 ±12.79)times/h〕,and the N3 sleep stage was on the lower limit of normal 〔(14.67 ±11.10)%〕. Compared with non-delirium group,the REM sleep was significantly decreased in delirium group〔(0.10±0.20)%vs.(8.83±3.81)%,t=4.782,P=0.001〕. Melatonin levels lost rhythm between day and night,and there was no difference in melatonin between delirium group and non-delirium group (time effect:F=1.370,P=0.287;between-group effect:F=1.646,P=0.250;interaction effect:F=1.558,P=0.247). The peak of melatonin levels of delirium group appeared on 06:00 〔(137.84±62.21)ng/L〕and 14:00 〔(148.24±58.8)ng/L〕, the minimum value on 22:00 〔(64.47 ±26.97) ng/L〕. But in non-delirium group,the peak of melatonin levels appeared on 02:00 〔(63.52 ±39.75)ng/L〕,the minimum value on 10:00 〔(44.87 ±11.19)ng/L〕. Conclusions ICU patients have sleep disorders,and the delirium patients have less REM stage. Normal rhythmic melatonin secretion changes of ICU patients were lost. The delirium peak of patients appears in the daytime.

Objective To investigate the expression of NLRP3 in different time point of HIBD neonatal rats and to search for critical time points and alleviate HIBD dysfunction.Methods 96 newborn rats of 7 days old were randomly divided into HIBD group(n=48) and Sham operation group(n=48).HIBD model was prepared by referring to Rice method.Brain tissue was taken after 6 h,24 h,72 h,7 d.Brain injury was detected by HE stain.The expression and distribution of NLRP3 and Caspase-1 were detected by immune fluorescence and Western blot,and IL-1β and IL-18 were detected by ELISA.Results HE staining and immunofluorescence showed that NLRP3 protein (HIBD group (0.63±0.07),Sham group(0.43±0.04)) was increased significantly since 6 h in HIBD group,and its downstream protein Caspase-1,IL-1β and IL-18 were successive activated.The results showed IL-1β (HIBD group(732.28± 108.42)pg/ml,Sham group(584.58± 36.35) pg/ml) was increased significantly since 6 h in HIBD group;Caspase-1 (HIBD group(0.67±0.09),Sham group(0.30±0.05)),IL-18 (HIBD group(683.84±31.83) pg/ml,Sham group(571.32±50.91) pg/ml) was increased significantly since 24 h in HIBD group(P＜0.05).Conclusion NLRP3 and its downstream inflammatory cytokines IL-1 β and IL-18 are up-regulated when HIBD occurs.The change of NLRP3protein expression in group HIBD is earlier than changes of neuron.NLRP3 signal may mediate and participate in the occurrence and development of HIBD.

BACKGROUND:Studies have shown that umbilical cord blood stem cel transplantation promote the recovery of spinal cord injury, and electroacupuncture also can inhibit the proliferation of astrocytes to reduce damage to scar formation, suggesting that a combination of umbilical cord blood stem cel transplantation and electroacupuncture may play an important role in the treatment of acute spinal cord injuries. OBJECTIVE:To observe the influence of transplantation of human umbilical cord blood stem cels combined with electroacupuncture at theDu channel on expression of nerve growth factor and neurotrophin 3 in rats with spinal cord injuries. METHODS: Seventy-two female Sprague-Dawlay rats were randomly divided into control group, injury group, transplantation group and combined therapy group. In the control group, only an incision on the back was sutured;in the injury group, a piece of saline-infiltrated gelatin sponge, 1 mm×2 mm×2 mm, was placed into the transected spinal cord at T10 level; in the transplantation group and combined therapy group, a piece of gelatin sponged infiltrated in the suspension of human umbilical cord blood stem cels was placed into the transected spinal cord, respectively, and then, electroacupuncture stimulation at the Duchannel was performed in the combined therapy group at 1 hour after modeling. Specimens were taken at 7, 14, 28 days after modeling in each group, and then immunohistochemistry, western blot and real time-PCR methods were used to detect the expression of nerve growth factor and neurotrophin 3. RESULTS AND CONCLUSION:Compared with the transplantation group, the expression of nerve growth factor and neurotrophin 3 was lower in the injury group but higher in the combined therapy group at 7, 14, 28 days after modeling (P < 0.05). The results of western blot and real time-PCR were consistent with those of immunohistochemical detection. Findings show that human umbilical cord blood stem cel transplantation combined with electroacupuncture has a remarkable synergistic effect in the treatment of spinal cord injury that can significantly up-regulate the expression of nerve growth factor and neurotrophin 3, and contribute to injured spinal cord repair, regeneration and functional recovery after spinal cord injury.

The establishment of clinical medicine with professional degree is an important reformation of high-level professionals in medical field. With the expansion of the number of postgraduates of clinical medicine with professional degree, how to establish and perfect the training mode and how to improve the training quality has become an important research for the postgraduate education with professional degree. This paper discusses the establishment and perfection of training mode of full-time postgraduates of clinical medicine with professional degree by combining the documents and development of professional degree in my university.

OBJECTIVE:To provide reference for the further research and development of bamboo juice. METHODS:Comb-ing the ancient and modern literatures,the processing historical evolution,origins,technology,chemical ingredients,quality stan-dards and efficacy were analyzed,the problems were found and solutions were put forward. RESULTS:Bamboo juice's processing can be traced back to the Liang Dynasty,still being used today with light bamboo juice. And now,varieties of bamboos(Phyllo-stachys glauca,Phyllostachys viridiglaucescens,Pleioblastus amarus,and Neosino calamus,etc.) were processed into medicine. The research for the chemical ingredients was mainly for the changes of bamboo juice with different varieties and amino acids,phe-nols,organic acids and inorganic elements in bamboo juice with different technologies,and there was no comprehensive study on the material basis of bamboo juice. Its quality standards must be improved. Although there was comparative study for the traditional processing technology and modern technology in chemical ingredients,the study did not compare the efficacy of its material founda-tions. The research for the efficacy of bamboo juice only involved relieving cough and resolving phlegm,and its relieving convul-sion,resuscitation,calming diabetes and other pharmacological effects haven't been reported. CONCLUSIONS:In the future, in-dept research can be carried out from the aspects of standardizing the name,base source and processing technology parameters, improving quality standards,strengthening basic research of effective substances,and improving traditional technology and equip-ment level.

Objective:To investigate the effect of homocysteine (Hcy) on cerebral perfusion and cognitive function in patients with arteriosclerotic cerebral small vessel disease (aCSVD).Methods:A total of 117 patients with aCSVD who visited the First Affiliated Hospital of Anhui Medical University from June 2020 to September 2022 were enrolled and divided into the aCSVD cognitive impairment group (aCSVD-CI, n=57) and aCSVD non-cognitive impairment group (aCSVD-NCI, n=60) according to the Montreal Cognitive Assessment score. Serum Hcy measurement, cognitive function assessment, and three-dimensional pseudo-continuous arterial spin labeling perfusion imaging scan were performed in all patients, and multivariate Logistic regression analysis was used to explore risk factors for cognitive impairment in patients with aCSVD. The cerebral blood flow and perfusion differential brain regions of the whole brain, grey matter, and white matter were compared between the two groups. Partial correlation analyses were performed between the serum Hcy, overall cognitive function scores and cerebral blood flow in grey matter, as well as between the cerebral blood flow in the perfusion differential brain area and cognitive function scores. The mediating effect model was used to analyze the role of grey matter blood flow in the relationship between serum Hcy and overall cognition. Results:The serum Hcy level in the CSVD-CI group was higher than that in the CSVD-NCI group [16.38(14.02, 18.58) μmol/L vs 14.40 (11.93, 15.73) μmol/L, Z=-3.81, P<0.001]. In terms of cerebral perfusion, compared with the aCSVD-NCI group, the aCSVD-CI group had significantly lower cerebral blood flow in grey matter ( Z=-3.22, P=0.001), left middle frontal gyrus ( t=-4.91, P<0.05), right middle frontal gyrus ( t=-5.14, P<0.05), and right orbital medial frontal lobe ( t=-4.38, P<0.05). In contrast, the left hippocampus ( t=4.58, P<0.05) had increased cerebral blood flow. Multivariate Logistic regression analysis showed that serum Hcy level was independent risk factor for cognitive impairment in aCSVD after controlling for multiple risk factors. Partial correlation analysis showed that left middle frontal gyrus blood flow ( r=-0.39, P=0.006), right middle frontal gyrus blood flow ( r=-0.44, P=0.002), and right orbital medial frontal lobe cerebral blood flow ( r=-0.43, P=0.002) were negatively correlated with the Stroop Color Word Test-C results. Left hippocampal cerebral blood flow was negatively correlated with Auditory Word Learning Test-long-delayed recall ( r=-0.43, P=0.002). Further mediation analysis showed that the effect of Hcy on cognitive function was partly mediated by grey matter cerebral blood flow (indirect effect=-0.11, P<0.001). Conclusion:Hcy is an independent risk factor for cognitive impairment in aCSVD, and part of the effect of elevated Hcy on cognitive impairment in aCSVD may be mediated by decreased gray matter cerebral perfusion.

OBJECTIVETo study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion.

METHODSHepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay.

RESULTSHepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness.

CONCLUSIONHepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.

Apoptosis ; Culture Media ; chemistry ; DNA, Mitochondrial ; genetics ; Ethidium ; chemistry ; Hep G2 Cells ; radiation effects ; Humans ; Radiation Tolerance ; genetics ; Sequence Deletion ; X-Rays