Objective To isolate and identify embryonic stem (ES) cells of rhesus monkey from mature blastocysts cultured in vitro . Methods Rhesus monkey blastocysts were obtained after in vitro maturation of oocytes, fertilization and maturation of early embryos. After the blastocysts were hatched naturally from the zone pellucida, the inner cell mass (ICM) was dislodged from blastocysts using the sealed end of a finely drawn Pasteur pipette and co cultured with the feeder cells. The ICM, resembling embryonic stem cell colony, was isolated, cultured, and identified. Results A total of 92 normal GV oocytes were obtained from 4 FSH prime rhesus monkeys. Six blastocysts with high quality were selected from 22 oocytes after culture in HECM 10 medium. One of the rhesus monkey ES cell lines, i.e. RS5 cell line, was finally isolated from 3 of the inner cell mass isolated from the 6 blastocysts. The RS5 cells presented a high nucleus/cytoplasm ratio, prominent nucleoli, and flatter colonies with individual and distinct cells. After successive passages for 5 months passage, the RS5 cells remained a normal karyotype, i.e. 42 of chromosomes and alkaline phosphatase (AP) positive, which meant the RS5 ES cell colony remained undifferentiated. After high density culture for a longer time, the ES cell could differentiate into multi types of cells. Conclusion The RS5 cell line has the ability to self renew and potential to differentiate. Thus, RS5 cell line belongs to embryonic stem cells.

Objective To investigate the mechanism of an antifibrotic drug, pirfenidone, in preventing radiation-induced pulmonary fibrosis. Methods Male BALB/C mice were randomized into 4 groups:control group (C), radiation alone group (R), pirfenidone alone group (P), and pirfenidone plus radiation group (P + R). Irradiation was administrated to the whole pulmonary with a single fraction of 12 Gy. The pirfenidone was given 0. 3 ml/kg/d from 3 days prior to irradiation to 12 weeks after.Bronchoalveolar lavage fluid (BALF) from right lung was collected for macrophages counting every monthmonthly until 6 months after irradiation, and left lungs were collected and fixed for. The pulmonary fibrosis was assessed by Masson trichrome staining. The plasma transforming growth factor β(TGF-β) was measured by ELISA. The lung hydroxyproline was evaluated by alkaline solution. Results Compared to group R, the counts of macrophages in BALF in group P + R were reduced by 76% and 62%, and hydroxyproline levels were reduced by 21% and 24% at the 4th and 5th months, respectively. The plasma TGF-β decreased from the 3rd month to 5th month. Pirfenidone markedly ameliorated the severity of lung fibrosis at the 4 - 6th month after radiation. Conclusions Pirfenidone can prevent radiation-induced pulmonary fibrosis, the mechanism of which may be the reduced of inflammation and collagen deposition by decreasing macrophages and hydroxyproline.

ObjectiveTo evaluate whether oral administration of Chinese tradiational medicine,Qing-Xue granula,can prevent mouse lung injury caused by thoracic radiation.Methods128 BalB/C mice were divided into 4 groups:control (C) group; radiation (R) group; radiation plus high dose Qing-Xue granula (H) group and radiation plus median dose Qing-Xue granula ( M ) group.The H and M groups were fed 0.64 g and 0.32 g of Qing-Xue granula dissolved in 0.5 nl anline once daily for two months,which were 4 and 2 times of human dosage,respectively.Whole thorax radiation of 12 Gy was delivered with a single ventral-dorsal field with 6 MV X-ray.Group C and group R received 21 days of 0.5 ml saline feeding.Mice were sacrificed at 1,2,4 or 6 months after radiation. Macrophage cell count of lung lavage fluid and hydroxyproline content of left lung were assayed,and the lung fibrosis was scorred according to the Ashcroft's criteria.The plasma interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) concentration were assayed with ELISA method.The One-way ANOVA was used to test the significance of any differences between groups at each time point. Results The macrophage cell number of lung lavage fluid was significantly lower in the 1st month in group M than in group R (2∶4,q =3.92,P ＜ 0.05 ),but had no significant difference between group M and C ( 1 ∶ 4,q =2.13,P＞0.05 ).The hydroxyproline content of group H was significantly lower than group R in the 1st and 6th months (q =3.62,3.54,all P ＜ 0.05 ),but still higher than group C ( q =4.09,3.72,all P ＜ 0.05 ).The fibrosis score of group H was significantly lower than group R in the 2nd,4th and 6th months (q=3.38 -4.16,all P＜0.05).The IL-6 concentration of group H was significantly lower than group R in the 1st month ( q=3.53,P＜0.05 ),but not significantly higher than group C (q =1.41,P＞0.05).The VEGF concentration was significantly higher in group R than group C since the 2nd month ( q =3.12 - 3.78,P ＜ 0.05 ).The VEGF concentration was significantly higher in group H and M than group R in the 2nd and 6th months ( q =3.08 - 3.92,all P ＜ 0.0 5 ).Conclusions Oral Chinese traditional medicine,Qing-Xue granula,could prevent radiation induced lung fibrosis in mice,especially at high dosage.The degree of elevation of VEGF in plasma was not parallel with that of lung fibrosis.

ObjectiveTo evaluate the effect on lung injury of gefitinib or/and radiation.Methods Totally 160 mice were divided into five groups:control (C) ;gefitinib (G) ;radiation (R) ;gefitinib followed by irradiation ( G + R) ;and R + G.12 Gy irradiation was delivered.Geiitinib fed by 200 mg/kg once daily for 3 weeks.Mice were sacrificed on 1,2,4 or 6 months after radiation.Macrophage count of lung lavage fluid and hydroxyproline assessed,lung fibrosis scored.and plasma TGF-β1 concentration assayed.One-way ANOVA was used to test the significance. Results The lung lavage macrophage cell number were significantly higher in group R,R + G and G + R than group C ( q =2.95 - 8.61,all P ＜ 0.05 ) on 4 and 6months,yet no significant difference between the three groups ( q =0.37 -3.49,all P ＜ 0.05 ) ; The macrophage was significantly lower in month 1,4 and 6 in group G than R,R + G and G + R ( q =3.37- 6.25,all P ＜ 0.05 ).The hydroxyproline content and the fibrosis score of G,R,R + G and G + R were significantly higher than C ( q =3.14 - 4.76,all P ＜ 0.05 ),but no significant difference between the four groups ( q =0.70 - 4.19,all P ＞ 0.05 ).The TGF-β1 concentration of R,G + R,R + G at all time points and TGF-β1 concentration of G at 1 st and 2nd months were significantly higher than C ( q =3.76 -8.09,all P ＜ 0.05).ConclusionsGefitinib could cause lung fibrosis in vivo in BalB/C mouse.The combination of gefitinib and radiation did not significantly exacerbate lung injury caused byeither alone.The mechanism of lung fibrosis caused by gefitinib might be different from that by radiation which needs further research.

Objective To evaluate the relationship between single nucleotide polymorphism(SNP) of candidate genes and radiation-induced esophagitis (RIE) in patients with lung cancer. Methods Between Jan. 2004 and Aug. 2006,170 patients with pathologically diagnosed lung cancer were enrolled in this study. The total target dose was 45-70 Gy( median 60 Gy). One hundred and thirty-two patients were treated with three-dimensional conformal radiotherapy(3DCRT) and 38 with two-dimensional radiotherapy(2DRT).Forty-one patients received radiotherapy alone, 78 received sequential chemoradiotherapy and 51 received concurrent chemoradiotherapy. Thirty-seven SNPs in 20 DNA repair genes were analyzed by using PCR-based restrieted fragment length polymorphism(RFLP). These genes were apoptosis and inflammatory cytoking genes including ATM, ERCC1, XRCC3, XRCC1, XPD, XPC, XPG, NBS1, STK15, ZNF350, ADPRT,TP53, FAS, FASL, CYP2D6 * 4, CASPASE8, COX2,TGF-β, CD14 and ACE. The endpoint was grade ≥2 R I E. Results Forty of the 170 patients developed grade ≥2 R I E, including 36 in grade 2 and 4 in grade 3. Univariate analysis revealed that radiation technique and concurrent chemoradiotherapy were statistically significant relatives to the incidence of R I E (P = 0. 032,0.049) , and both of them had the trend associating with the esophagitis( P = 0.072,0. 094 ). An increased incidence of esophagitis was observed associating with the TGF-β1-509T and XPD 751 Lys/Lys genotypes ( χ2 = 5.65, P = 0.017 ;χ2 = 3.84, P = 0. 048 )in multivariate analysis. Conclusions Genetic polymorphisms in TGF-β1 gene and XPD gene have a significant association with radiation-induced esophagitis.

Many human genetic diseases, including Hutchinson-Gilford progeria syndrome (HGPS), are caused by single point mutations. HGPS is a rare disorder that causes premature aging and is usually caused by a de novo point mutation in the LMNA gene. Base editors (BEs) composed of a cytidine deaminase fused to CRISPR/Cas9 nickase are highly efficient at inducing C to T base conversions in a programmable manner and can be used to generate animal disease models with single amino-acid substitutions. Here, we generated the first HGPS monkey model by delivering a BE mRNA and guide RNA (gRNA) targeting the LMNA gene via microinjection into monkey zygotes. Five out of six newborn monkeys carried the mutation specifically at the target site. HGPS monkeys expressed the toxic form of lamin A, progerin, and recapitulated the typical HGPS phenotypes including growth retardation, bone alterations, and vascular abnormalities. Thus, this monkey model genetically and clinically mimics HGPS in humans, demonstrating that the BE system can efficiently and accurately generate patient-specific disease models in non-human primates.
Animals ; Disease Models, Animal ; Female ; Gene Editing ; Humans ; Lamin Type A/metabolism* ; Macaca fascicularis ; Progeria/pathology*