To optimize the culture environment and protocol of hematopoietic cells' expansion, avoiding the fluctuation caused by medium changing in stirred culture and concentration gradient in static culture, the hematopoietic cells from cord blood (CB) were cultured in a stirred bioreactor connected with a cell retention system, which is a gravity sedimentation settler designed for hematopoietic cell. Total cells expanded 11.5 and 18.6 fold respectively in the twice perfusion stirred cultures, in which CFU-Mix was expanded 23.2 and 20.4 fold, CFU-GM 13.9 fold and 21.5 fold, BFU-E 8.0 fold and 6.9 fold, CD34+ cells 17.1 fold and 15.4 fold. After 12-day culture, it was obtained that 1082 x 10(6) total cells, 6.31 x 10(6) CFU-GM, 6.2 x 10(6) CFU-Mix and 23 x 10(6) CD34+ cells from 267 x 10(6) CB mononuclear cells (MNC) in the first culture, and 1080 x 10(6) total cells, 4.65 x 10(6) CFU-GM, 11.0 x 10(6) CFU-Mix, and 25.0 x 10(6) CD34+ cells from 180 x 10(6) CB MNC. These two cultures met to the clinical scale. Due to the optimized dissolved oxygen (DO) and stable culture environment, the rate of stem/progenitor cells to total cells in the perfusion culture was higher than that in T-flask cell-retention feeding culture. But the cell growth was inhibited in the later phase of perfusion culture, when the cell density is high. The inhibition should be attribute to the high cell density itself. The perfusion culture environment in bioreactor with optimal DO and pH controlling is more favorable for stem/progenitor cells' maintenance and expansion, and the expanded cells' number has reached a clinical scale. But the high cell density in the later phase of perfusion culture caused inhibition to mature hematopoietic cell's growth.
Bioreactors ; Cell Culture Techniques ; methods ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans

OBJECTIVETo study the methods of three-dimensional reconstruction of digitized virtual hepatic.

METHODSImages of DSCF 2511-2520 were taken from the database of digitized Virtual Chinese Human female No. 1 (VCH-F1). Method of insertion value algorithm of three-dimensional reconstruction was used to make three-dimensional block diagram. In ordering to auto-judge the position of hepatic solid and hepatic ducts, these images were shown with different colors according to the character of color and location of every spot.

RESULTSStereo image of hepatic solid could be shown satisfactorily. Every shape of stereo image and the structure of hepatic duct could be shown by revolving the three-dimensional image with different direction.

CONCLUSIONSThe image of hepatic database of digitized Virtual Chinese female No. 1 was exact. The three-dimensional image of the liver and hepatic duct made by insertion value algorithm of three-dimensional reconstruction were distinct, and it was a ideal method of three-dimensional reconstruction.

The function of immune system degenerates in an aging-dependent manner and this results in immunosenescence. Human immune system includes two parts: genetic/innate immunity and adaptive immunity. The former is involved in monocytes, nature killer cells, and dendritic cells, the later is involved in acquired B and T lymphocytes. During the aging of immunity system, the both parts of immunity are damaged to some degree. Generally, innate immunity seems well-retained and the acquired immunity is degenerative seriously with aging. Immunocyte senescence is closely related to the elderly decreased ability to control infectious disease, cancer and to their generally poor response to vaccination. This review summarized the research progress on immunosenescence characteristics in aged phase.
Age Factors ; Aging ; immunology ; Antibody Formation ; immunology ; Cellular Senescence ; Humans ; Immunity, Cellular ; immunology ; Lymphocyte Activation

This study was purposed to evaluate the safety and curative effect of autologous cytokine induced killer cells (CIK) combined with low-dose IL-2 regimen containing immune enhancement of thymic peptide on elderly patients with B-cell chronic lymphocytic leukemia (B-CLL). Thymic peptide α1 was subcutaneously given as the immunoenhancement agent at a dose of 1.6 mg/d, 14 days as one cycle. Peripheral blood mononuclear cells (PBMNC) from 5 patients with B-CLL were isolated once a week to induce ex vivo CIK cells through culture in the context of interferon (IFN)-γ, interleukin (IL)-2 and anti-CD3 monoclonal antibody. The PBMNC were separated from patients before and after 14 days as one cycle of thymic peptide α1 administration. Parameters of amplification ability, effector cells quantity, lymphocyte subgroups percentage and antitumor cytotoxicity were compared before and after thymic peptide administration. The 5 patients were treated with CIK cells combined with low-dose IL-2 regimen immediately after injection of thymic peptide α1. The CIK cells plus low-dose IL-2 regimen containing thymic peptide enhancement was defined as: thymic peptide α1 1.6 mg/d was subcutaneously administered once every other day; (4 - 6) ×10(9) of CIK cells were transfused followed by IL-2 subcutaneous administration of 1 mU/d on days 1-10, 28 days as one cycle. Clinical evaluation parameters including cellular immunity function, CLL related biomarkers, disease state and infectious frequency and degree were investigated before and after CIK cells infusion puls IL-2. The results showed that the amount of amplified CIK cells, the percentage and amplification times of effector cells and antitumor cytotoxicity more significantly increased after thymic peptide α1 treatment than before its use (P < 0.05). The total 46 cycles of CIK cells infusion plus IL-2 were completed in the 5 CLL patients. No adverse reaction was observed. After treatment of CIK cells plus IL-2, the general conditions of 5 CLL patients were to different extent improved. Simultaneously, percentages of CD3(+), CD3(+)CD8(+), and CD3(+)CD56(+) cells in peripheral blood remarked by raised (P < 0.05), the serum level of β2 microglobulin was significantly declined (P < 0.05), and the frequency and degree of infection was also decreased (P < 0.05). Following CIK cells plus IL-2 therapy, the transformation of disease state from partial remission (PR) to complete remission was seen in 3 patients, from stable disease (SD) to PR in 1 patient, and from progress of disease to SD in 1 patient. It is concluded that the regimen of autologous CIK cells combined with low-dose IL-2 containing immune enhancement of thymic peptide is safety and effective for the treatment of elderly patients with B-CLL.
Aged ; Aged, 80 and over ; Cytokine-Induced Killer Cells ; immunology ; Humans ; Interleukin-2 ; administration & dosage ; therapeutic use ; Leukemia, Lymphocytic, Chronic, B-Cell ; therapy ; Male ; Thymosin ; immunology

AIM: To observe the growth status and morphological changes of primary cultured bulbar conjunctival fibroblasts in different stages of conjunctivochalasis(CCH), and to determine the best passage time, so as to obtain stable and consistent CCH bulbar conjunctival fibroblasts.METHODS: CCH primary bulbar conjunctival fibroblasts were obtained by tissue block adhesion method. The fibroblasts were purified by trypsin differential digestion method. The growth status and morphological changes of fibroblasts in different periods were observed and recorded under inverted microscope. The fibroblasts were identified by immunofluorescence cytochemical staining.RESULTS: After 24h of CCH conjunctival tissue adherent to the wall, a small number of cells would be seen crawling out around the tissue blocks. The logarithmic phase of cell growth was from the 2-7d. The cells grew fast, with vigorously proliferation, clear outline, uniform distribution, increas in numbers and clear nuclei. From the 9-15d, the cell growth entered the plateau stage, the tissue blocks gradually aged and lost activity. The cells grew slowly, arranged loosely, the volume became larger, the shape became flat, and a large number of granular substances and vesicles were produced in the cytoplasm. Some cells fell off from the bottom of the culture bottle, and large gaps appeared between the cells. After subculture and purification, the size and morphology of fibroblasts were basically the same. Through cell identification, fibroblasts were long spindle, flat star or multi-process spindle, wide in the middle, oval nucleus, relatively small at both ends, with 2-3 slender processes of different lengths extending outward.CONCLUSION: Primary CCH bulbar conjunctival fibroblasts can be successfully obtained by tissue block adhesion method. When the cells grow to the 8d, they can be digested and passaged to obtain stable and consistent CCH conjunctival fibroblasts.

The chemotaxis response of Erwinia carotovora to different sugars and amino acids in four kinds of chemotactic parameters (concentration, time, temperature and pH ) was determined by capillary method. The results showed that when pH was 8, concentration was 0.025 mg•L ⁻¹, culture temperature was 25 ℃ and the duration was 60 minutes, the optimal chemotaxis rate of lysine was 2.509,when pH was 6, concentration was 0.25 mg•L ⁻¹, culture temperature was 25 ℃ and the duration was 60 minutes, the optimal chemotaxis rate of arginine was 2.218 8,when pH was 7, concentration was 0.25 mg•L ⁻¹, culture temperature was 30 ℃ and the duration was 60 minutes, the optimal chemotaxis rate of L-rhamnose was 3.091 2, when pH was 6, concentration was 0.25 mg•L ⁻¹, culture temperature was 30 ℃ and the duration was 45 minutes, the optimal chemotaxis rate of D-arabinose was 3.026 3. Sugars and amino acids had obvious chemotaxis with E. carotovora,the high concentration of carbohydrate and amino acid exited an inhibitory effect on chemotaxis response of E. carotovora, and the chemotaxis response decreased with the increase of concentration of carbohydrates and amino acids.

Aim To clarify the effect of circulating exosomes on hypertension, screen out miRNAs which plays a key role, and explore its function.Methods The plasma exosomes of spontaneously hypertensive rats were extracted and injected into Sprague Dawley rats.The blood pressure changes of rats were detected.Plasma exosomes and exosomal RNA of hypertensive patients and SHR were extracted.Real time PCR was used to verify the expression changes of the selected 8 miRNAs; Western blot was used to detect the expression changes of LKB1 and PTEN protein levels in human umbilical vein endothelial cells transfected with miR-17-5p mimics.Results The plasma exosomes of SHRs significantly increased the blood pressure of SD rats(P<0.05).The expression of miR-17-5p and miR-218-5p in the plasma exosomes of hypertensive patients and SHRs both significantly increased.miR-17-5p inhibitors significantly attenuated the effect of SHR-exos on raising blood pressure.miR-17-5p mimics down-regulated the expression of LKB1 and PTEN in HUVECs cultured in vitro.Conclusions The plasma exosomes of SHR can significantly increase blood pressure of Sprague Dawley rats.miR-17-5p may be the key miRNA.exo-miR-17-5p may promote the occurrence and development of hypertension by regulating the LKB1/PTEN signal.

Aim To investigate the slope ratio method for the determination of anticoagulant activity of leeches. Methods Three batches of leeches, four groups of Japanese medical vermiculite yinpian and fifteen groups of leech preparations were chosen, with contrast medicinal leeches herbs and Philippine cattle leech contrast medicinal materials, and different concentrations of leaching solutions were prepared in parallel. APTT value was determined after anticoagulant activity was determined by slope ratio method for the joint validation of laboratory, intermediate precision and accuracy between the linear range. Results The slope ratio method was accurate and accurate in the determination of anticoagulant activity of leeches, with linearity between 64% and 156% relative titer level. Conclusion Slope ratio method can be used to determine the anticoagulant activity of leeches.

Air Pollution ; Asthma/drug therapy* ; China ; Humans ; Treatment Adherence and Compliance

The purpose of this study was to explore the clinicopathological features, therapy and prognostic factors of elderly patients with non-Hodgkin's lymphoma (NHL). The clinical data including general clinical characteristics, pathological features, chemotherapy selection and treatment response of 30 patients with NHL in our hospital from January 2003 to December 2012 were analyzed retrospectively. The survival was analyzed by using Kaplan-Meier methods, and the prognosis was evaluated by COX regression multivariate analysis model. The clinical parameters selected include age, Ann Arbor stage, international prognostic index (IPI), B symptom and lactate dehydrogenase (LDH) levels. The results showed that all the patients suffered from underlying disease, and the cardiovascular disease (hypertension, coronary heart disease, arrhythmia) is the most common, and minority (8/30) combined with secondary tumor, the 63% (19/30) cases had B symptoms at diagnosis. only 2 cases were diagnosed as T-cell lymphoma; the 93% (28/30) cases combined with B-cell lymphoma, 57% (17/28) of them combined with diffuse large B-cell lymphoma. Ann-Arbor stage ≤ IIwas 37% (11/30);10(37%) patient's IPI score was ≤ 2, and 67% (20/30) was scored 3-5; 13(43%) patient's serum LDH level was abnormal. Modified R-CHOP chemotherapy was given individually on the basis of clinical features. The patients achieved complete remission, partial remission, stable disease, or progressive disease accounted for 14 (46.7%), 13 (43.3%), 1 (3.3%), and 2 (6.7%), respectively; the total reaction rate was 90% after 4 cycles of chemotherapy; the overall survival (OS) rate at 1 and 2 years was 73.3% and 43.3%, and progression-free survival (PFS)rate at 0.5 and 1 years was 62.2% and 54.9%; multivariate analysis by COX regression showed that B symptoms and Ann-Arbor stage were independent factors (P = 0.014, 0.039; RR = 6.678, 4.939, respectively) affecting the OS of elderly NHL, and IPI score affected PFS independently. It is concluded that elderly patients with NHL usually are of late stage at newly diagnosis and have suffered from underlaying diseases. Besides strengthening supportive treatment, modified R-CHOP chemotherapy should be given individually according to different prognosis. B symptoms and Ann-Arbor stage >II are indicators for poor prognosis of elderly NHL.
Aged ; Aged, 80 and over ; Female ; Humans ; Lymphoma, Non-Hodgkin ; diagnosis ; pathology ; therapy ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Treatment Outcome