The purpose of this study was to transplant neonatal rat with human cord blood Lin(-) cells to test the possibility of this xenograft model. The Lin(-) cells were purified from human cord blood (CB) using negative selection strategy based on different lineage-specific antigens. The Lin(-) cells were injected into the liver of neonatal rats using a microinjector at an average of 5 x 10(5) cells for each. Peripheral blood (PB) and spleen were collected at 2,4 and 8 weeks after injection. Flow cytometry was performed to detect human cells in the rat PB, PCR was used to detect human cells in PB as well as spleen. The results showed that a definite proportion of human cells existed in peripheral blood of chimeric rat and the human specific beta2 microglobulin gene fragments were detected in spleen genomic DNA of chimeric rat. It is concluded that human/rat chimera model can be developed with neonatal rats. Human/rat xenograft model may provide a useful and convenient method for human hematopoietic stem cell assay in vivo.
Animals ; Animals, Newborn ; Cord Blood Stem Cell Transplantation ; DNA ; genetics ; Flow Cytometry ; Humans ; Leukocyte Common Antigens ; blood ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Spleen ; metabolism ; Transplantation Chimera ; blood ; genetics ; immunology ; Transplantation, Heterologous ; beta 2-Microglobulin ; genetics

AIMTo investigate the dynamic changes of Egr-1 expression in the lungs of acute pulmonary embolism of rats by infusion of autoblood thrombs.

METHODSThe model of pulmonary embolism by infusion of autoblood thrombs in the pulmonary artery of rats was established and the mean pulmonary arterial pressure was continuously monitored by computer, and the results were evaluated by lung perfusion scan and pathological changes. Expression of Egr-1 proteinum and mRNA were measured by immunohistochemistry and reverse transcription polymerase chain reaction.

RESULTSThe mPAP of rats was increased significantly after infusion of autoblood thrombs at the half hour, and reached high level at the second hour, then remained the high level to four hours compared with group control at the same time point (P < 0.01). ECT image was showed significantly filling defect after infusion of autoblood thrombs at the first hour. The infused thromb was witnessed by hematoxylin and eosin stain. In the tracheal epithelium cells, alveolar epithelium cells and vascular smooth muscle cells of embolism rats, Egr-1 protein expression was increased significantly after embolization at the second hour compared with group control at the same time point (P<0.01), and was decreased slowly at the fourth hour. Egr-1 mRNA expression was showed the similar changes.

CONCLUSIONExpression of Egr-1 was low level in group control, but increased significantly after infusion of autoblood thromb at the second hour in the specificity of cells, suggesting that Egr-1 expression might be an important link of pathological changes in the acute pulmonary embolism.

Animals ; Early Growth Response Protein 1 ; genetics ; metabolism ; Gene Expression ; Lung ; metabolism ; Male ; Pulmonary Embolism ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

Bone marrow mesenchymal stem cells (MSCs) are multipotent tissue stem cells that can be induced in vitro to differentiate into a variety of cells such as osteoblasts, chondrocytes and adipocytes. MSCs are useful vehicles for both cell and gene therapy for a variety of diseases. Here, we injected human MSCs with enhanced green fluorescent protein (EGFP) into the striatum of Parkinson disease (PD) rat and examined their survival, migration, differentiation, and the behavior changes in PD rats, which will provide a theoretical foundation and technical method for clinic PD therapy by stem cells. The results showed that human bone marrow MSCs can survive in rat brain for a long time (exceeding 70 d). MSCs were found in multiple areas of the rat brain including the striatum, the corpus callosum, contralateral cortex and even the brain vascular wall. Immunocytochemical staining suggested that implanted cells expressed human neurofilament (NF), neuron-specific enolase (NSE) and glial fibrillary acid protein (GFAP). At the same time, remission in abnormal behavior of the PD rats appeared. Rotation scores decreased gradually from 8.86+/-2.09 r/min pre-transplantation to 4.87+/-2.06 r/min 90 d post-transplantation (statistic result showed P<0.05).
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Movement ; Corpus Striatum ; Green Fluorescent Proteins ; administration & dosage ; Humans ; Male ; Mesenchymal Stromal Cells ; cytology ; Parkinson Disease ; therapy ; Rats ; Rats, Wistar ; Stem Cell Transplantation ; methods ; Transplantation, Heterologous

To investigate the expression of telomerase in cord blood hematopoietic stem/progenitor cells during their committed differentiation in vitro and provide an index of monitoring the proliferating potential of the hematopoietic stem/progenitor cells and security for clinical application. Human CD34 positive cells were isolated from umbilical cord blood by using magnetic cell sorting system (MACS), and were induced to differentiation with hematopoietic growth factors (SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO) in a liquid culture system. The telomerase activity and the cytalytic subunit of telomerase (hTERT) of the cells were analysed during different periods of culture by using TRAP-PCR, TRAP-ELISA, Western blot and RT-PCR techniques, respectively. The results showed that a peak of cell growth was achieved on day 14 - 21 during induction of differentiation in vitro. Total cell number could increase 1006.4 +/- 103.2 times and could not increase there after. Telomerase activity and hTERT expression were low in freshly isolated cord blood CD34(+) cells and increased after about 7 days of culture in addition of cytokine combinations of SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO, respectively. The telomerase activity and hTERT decreased after 14 days of culture and were not detected after 28 days of culture. It was concluded that the hematopoietic stem/progenitor cells can be expanded in large number in vitro and do not have the character of immortality and the telomerase activity could be a useful index in hematopoiesis regulation.
Antigens, CD34 ; blood ; Blotting, Western ; Cell Differentiation ; DNA-Binding Proteins ; Enzyme-Linked Immunosorbent Assay ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; enzymology ; Humans ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Telomerase ; genetics ; metabolism

To evaluated the feasibility of preventing infection after high dose chemotherapy and radiotherapy using the granulocytes derived from differentiated from hematopoietic stem/progenitor cells ex vivo, human CD34-positive cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS), and the cells committedly differentiated with hematopoietic cytokines (SCF + IL-3 + IL-6 + G-CSF) in a liquid culture system. The expanded cell number, ratio of the viable cells, chromosome and phenotype of the differentiated cells and safety analysis of expanded cells were detected by using cell count, trypan blue exclusion test, karyotype analysis, flow cytometry and tumorigenic model of nude mice, respectively. The results showed that the combination of cytokines increased cell number by (1006.4 +/- 103.2) folds and flow cytometric analysis showed myeloid marker CD11b expressed in the about 60% cells. The growth peak of differentiated cells was at 14 days of culture and decreased at about 33 days. No abnormality was found in the karyotype analysis of expanded cells. No tumor was found in the nude mice injected with expanded cells after 35 days and the expanded cells had the ability of phagocytizing bacteria. It is concluded that the cells, differentiated from CD34(+) cells, expanded ex vivo possess the function of granulocyte and it was safe for clinical trial.
Animals ; Antigens, CD34 ; analysis ; Cell Differentiation ; Fetal Blood ; cytology ; Granulocytes ; cytology ; immunology ; Hematopoietic Stem Cells ; cytology ; Humans ; Karyotyping ; Mice ; Mice, Inbred BALB C ; Phagocytosis

BACKGROUNDConventional high ligation and stripping of the great saphenous vein (GSV) has a good curative effect but is highly traumatic with a considerable relapse rate. Subfascial endoscopic perforator surgery (SEPS) plus endovenous laser treatment (EVLT) could be applied as individual therapy. This study aimed to evaluate the feasibility of performing combined SEPS and EVLT without impacting GSV in the management of valvular insufficiency of the lower-limb venous perforators.

METHODSPlacement of lower-limb venous perforator insufficiency was marked by ascending phlebography in 83 affected limbs from September 2010 to June 2011. After randomization, SEPS was performed on 41 limbs to address the insufficiency of the venous perforators under the deep fascia, in combination with EVLT to close the superficial varicose veins without impacting the GSV. The remaining 42 limbs were treated using traditional GSV phlebectomy as controls.

RESULTSPostoperatively, all varicose veins were resolved, with lightening of the pigmentation and healing of the ulcer. Within a follow-up period of 5 - 11 months, no symptoms had recurred. Compared with the control group, the operation time, the number of incisions sutured, and the in-hospital time decreased on average by 1.5 hours, 4.7, and 6.8 days, respectively (P < 0.01 in all cases).

CONCLUSIONCombined SEPS and EVLT for treatment of valvular insufficiency of the lower-limb venous perforators offer the advantages of microtrauma and rapid cure.

Adult ; Aged ; Endovascular Procedures ; methods ; Female ; Humans ; Male ; Middle Aged ; Saphenous Vein ; surgery ; Varicose Ulcer ; surgery ; Varicose Veins ; surgery ; Vascular Surgical Procedures ; methods

OBJECTIVETo establish a long-term culture system for mouse spermatogonial stem cells (SSCs) and to discuss the key factor that supports mouse SSC self-renewal and proliferation.

METHODSTestis cells from 4-6 days postpartum male transgenic BALB/c mce were collected by a modified two-step enzymatic digestion method and plated on 0. 2% elatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml basic fibroblast growth factor (bFGF) and 200 ng/ml GDNF-family receptor alpha 1 (GFRalpha1) were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis.

RESULTSAfter 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months. Immunocytochemical staining showed that Oct4 was specifically expressed in the cultured SSC nucleus and GFRalpha1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia.

CONCLUSIONSSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.

Animals ; Cell Culture Techniques ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Spermatogonia ; cytology ; Stem Cells ; cytology

Objective The level of lactic acid in blood can reflect the degree of ischemia and hypoxia of brain tissue and cerebral perfusion pressure. The aim of this paper is to explore the value of blood lactate and lactate clearance in evaluating the survival rate and neurological outcome of patients with craniocerebral trauma. Methods The clinical data of 497 craniocerebral trauma patients admitted to our hospital from September 2017 to July 2018 were collected and retrospectively analyzed. Patients were divided into groups with different 6 h lactate clearance rates and admission lactate levels, and the differences in mortality and outcome of neurological function in each group were compared. Results The serum admission lactate levels、serum lactate levels at 6 hours, 28-day mortality and 28-day poor nerve function prognosis rate of patients with different 6h lactate clearance rates were statistically significant differences(P < 0. 05). The efficacy of 6h lactic acid to predict the mortality rate of patients was better than that of admission lactic acid and 6h lactate clearance rate (Z=3.71、Z=3.95,P<0.05). However, in predicting the neurological function of patients, the lactate clearance rate is not better than blood lactate level at any time(Z=1.30，Z=0.81，P>0.05). Conclusion 6h lactic acid has the best ability to judge the mortality of patients while lactic acid clearance rate is not better than the blood lactate level at any time in predicting the neurological function of patients.

Alzheimer's disease(AD) patients in China have been surging, and the resultant medical burden and care demand have a huge impact on the development of individuals, families, and the society. The active component compound of Epimedii Folium, Astragali Radix, and Puerariae Lobatae Radix(YHG) can regulate the expression of iron metabolism-related proteins to inhibit brain iron overload and relieve hypofunction of central nervous system in AD patients. Hepcidin is an important target regulating iron metabolism. This study investigated the effect of YHG on the expression of a disintegrin and metalloprotease-17(ADAM17), a key enzyme in the hydrolysis of β amyloid precursor protein(APP) in HT22 cells, by mediating hepcidin. To be specific, HT22 cells were cultured in vitro, followed by liposome-mediated siRNA transfection to silence the expression of hepcidin. Real-time PCR and Western blot were performed to examine the silencing result and the effect of YHG on hepcidin in AD cell model. HT22 cells were randomized into 7 groups: control group, Aβ25-35 induction(Aβ) group, hepcidin-siRNA(siRNA) group, Aβ25-35 + hepcidin-siRNA(Aβ + siRNA) group, Aβ25-35+YHG(Aβ+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aβ25-35+hepcidin-siRNA+YHG(Aβ+siRNA+YHG) group. The expression of ADAM17 mRNA in cells was detected by real-time PCR, and the expression of ADAM17 protein by immunofluorescence and Western blot. Immunofluorescence showed that the ADAM17 protein expression was lower in the Aβ group, siRNA group, and Aβ+siRNA group than in the control group(P<0.05) and the expression was lower in the Aβ+siRNA group(P<0.05) and higher in the Aβ+YHG group(P<0.05) than in the Aβ group. Moreover, the ADAM17 protein expression was lower in the Aβ+siRNA group(P<0.05) and higher in the siRNA+YHG group(P< 0.05) than in the siRNA group. The expression was higher in the Aβ+siRNA+YHG group than in the Aβ+siRNA group(P<0.05). The results of Western blot and real-time PCR were consistent with those of immunofluorescence. The experiment showed that YHG induced hepcidin to up-regulate the expression of ADAM17 in AD cell model and promote the activation of non-starch metabolic pathways, which might be the internal mechanism of YHG in preventing and treating AD.
ADAM17 Protein ; Alzheimer Disease/genetics* ; Amyloid beta-Peptides ; Drugs, Chinese Herbal/pharmacology* ; Hepcidins/genetics* ; Humans ; Pueraria

Temporary prescription preparation is the preparation processed into different dosage forms by relevant pharmacist according to the temporary preparation requirement and the personalized prescription made by the doctor in accordance with the syndrome differentiation and drug performance.It is an important part in personalized pharmaceutical services.Rational design of process route,production equipment and quality control method for the temporary prescription preparation,and establishment of technology research strategy and mode in accordance with the characteristics of traditional Chinese medicine temporary prescription preparations play an important role in promoting the development of the temporary prescription preparations.To promote the normalization,standardization and intelligent development of temporary prescription preparations,we would comprehensively summarize the significance,policy,technology characteristics,technology research status quo and existing problems in this paper,and put forward the research direction of temporary prescription preparation technology based on the physical properties of raw materials,equipment research strategy,and intelligent manufacturing technology.Thus it will push the inheritance and innovation of temporary prescription preparation.
Medicine, Chinese Traditional ; Prescriptions ; standards ; Quality Control ; Research Design