Using the character of natural aggregation of CHO cells, and an ultrasonic and sedimentation column combined perfusion system to promote cells aggregation and retention into bioreactor,recombinant CHO cell strain MK3-A2 was cultured,which could secrete rhTNK-tPA, by a serum-free perfusion culture system. The culture periods in this two experiments were as long as 77 and 110 days respectively. The cells density reached 2?107 cells /ml. The average volumetric productivity of rhTNK-tPA was 89 mg/L?d, and the highest one was 216mg/L?d.The cells aggregation rate was approximately 90%, and the diameters of most of them were 285～570?m. During the perfusion culture the cells retention rate almost kept in 95% and the viability of cells was more than 85%.Thus, it means that aggregation culture with such perfusion system could be used to scale up produce biopharmaceuticals instead of microcarrier culture system.

OBJECTIVETo explore function and related molecular mechanism of osteopractic total flavone (OTF) on tendon healing in rats.

METHODSTen male rats aged for 8 weeks were collected and weighted from 180 to 220 g. Tendon stem cells were cultivated, the third tendon stem cells were used for experiment. OTP treated with 0, 0.1, 1, 10 ng/ml were added into tendon stem cells, and expression change of ALP, Runx2, OCN, VEGF, P-S6, P-4E/BP1 were detected after 14 days. Forty male rats aged for 8 weeks (weighted 180 to 220 g) were established extra-articular tendon-bone transplanting healing model, and divided into experimental group and control group. Experimental group were treated with OTF(100 mg·kg⁻¹·d⁻¹), while control group was treated by normal saline with the same volume. Tendon-bone healing degree were detected by biomechanical testing at 3 and 6 weeks after surgery, histological detection were applied to detect tendon-bone healing and number of new vessles.

RESULTSAfter treated by OTP, ALP staining and active index detection showed there were statistical differences among 0, 0.1, 1, 10 ng/ml group. After 14 days' cultivation, western blotting results showed mTOR downstream marker protein P-S6 protein expression were gradually increased with increase of density of OTP, expression of P-4E/BP1 was reduced, while expression of Runx2, OCN, VEGF were increased. Biological detection results showed that there was no significant difference in mechanical strength between experimental group(0.78±0.05) N/mm and control group (0.51±0.02) N/mm at 3 weeks after surgery, while mechanical strength in experimental group (1.36±0.09) N/mm was higher than control group (1.01±0.08) N/mm at 6 weeks after surgery. Histological results showed maturity of tendon-bone surface cell were higher at 3 and 6 weeks in experimental group, sharpey fiber growth more density, calcification extent of mesenchyme was high, and new bone, vessels were increased.

CONCLUSIONSOTF could promote osteogenic differentiation of tendon stem cells through mTOR signaling in vitro, and stimulate tendon-bone healing in bone tunnel and enhance connection quality between tendon and bone.

Animals ; Biomechanical Phenomena ; Bone Transplantation ; Cell Differentiation ; Cells, Cultured ; Flavones ; pharmacology ; Male ; Osteogenesis ; Rats ; Stem Cells ; cytology ; TOR Serine-Threonine Kinases ; metabolism ; Tendons ; cytology ; transplantation ; Wound Healing

This study was purposed to evaluate the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on hematopoietic reconstruction and survival in beagles exposed to mixed fission neutron and γ-ray. 13 beagles were unilaterally exposed to single dose of 2.3 Gy 90% neutrons. The experiments were divided into 3 groups: irradiation control group (no any treatment, n = 4), supportive care group (n = 5) and rhG-CSF plus supportive care group (n = 4, abbreviated as rhG-CSF group) in which the beagles were subcutaneously injected with 200 µg/kg of rhG-CSF early at half an hour and 24 hours post-irradiation respectively. The results showed that 2.3 Gy 90% neutron irradiation induced a severe acute radiation sickness of bone marrow type. The administration of rhG-CSF increased the survival rate from 60% in supportive care group to 100%. Twice injection of rhG-CSF in the first 24 hours reduced duration of neutropenia, enhanced neutrophil nadir and promoted neutrophil recovery when compared with control cohort administered clinical support. The number of colony-forming cells (CFU-GM, CFU-E, and BFU-E) in peripheral blood of rhG-CSF treated canines increased 2-to 5-fold relative to those of the supportive care group on day 3. All canines treated with rhG-CSF achieved hematopoietic reconstruction as evidenced by the pathological section of sternum while severe shortage of hemopoietic cells remained in the cohorts given supportive care alone. It is concluded that the combination of supportive care and high-dose rhG-CSF can accelerate hematopoietic recovery and enhance survival of dogs exposed to 2.3 Gy mixed neutron and gamma ray.
Animals ; Dogs ; Gamma Rays ; adverse effects ; Granulocyte Colony-Stimulating Factor ; administration & dosage ; pharmacology ; Hematopoietic System ; drug effects ; radiation effects ; Neutron Diffraction ; Recombinant Proteins ; administration & dosage ; pharmacology ; Survival Rate