Mek 7222k

On day 17 after surgery, tail tip blood and whole blood from the ophthalmic vein were collected from the mice before euthanasia; serum was also obtained after blood coagulation. Blood glucose level was measured using a blood glucose meter (ACCU-CHEK; Roche Diabetes Care GmbH, Mannheim, Germany) after starving the mice for 12 h. All blood routine tests, including the determination of white blood cell count (WBC), haemoglobin (Hb) level, red blood cell count (RBC), haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH) level, mean corpuscular volume (MCV), red blood cell distribution width coefficient of variation (RDW-CV), platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW), and platelet haematocrit (PCT), were performed using an automatic blood cell analyser (MEK-7222K; NIHON KOHDEN CORP., Japan). Additionally, biochemical parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), blood urea nitrogen (BUN), and serum creatinine (Scr), were measured using an automatic biochemical analyser (AU480 Chemistry Analyser; Beckman Coulter, Brea, CA, USA).

To further evaluate whether the secreted BMP-2 protein can cause toxicity in vivo, blood, liver, spleen, and kidney samples were collected from the control group and the treated group. Eighteen male Wistar rats (average weight 350g) were divided into two groups: the cell sheet (without gene modification) (CS) group as a control group and the BMP/CS treatment group. A single defect was prepared on one side of the midsagittal suture at the dorsal part of the parietal region, then the cell sheet from the control group or the BMP-treated group was transplanted to the defect. The surgical procedure was the same as described previously. At 1, 3, and 7 days after surgery, blood samples and serum samples were collected. Liver, spleen, and kidney samples were obtained on day 7 after surgery. The hematological parameters (white blood cell number, red blood cell number, hemoglobin concentration, and platelets) were measured by a hematology analyzer (MEK-7222K; Nihon Kohden, Tokyo, Japan). Serum biochemical parameters of total protein, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine were determined using commercial kits (Roche Diagnostics, Mannheim, Germany). Liver, spleen, and kidney samples were prepared for histologic analysis. They were fixed in 4% paraformaldehyde solution, then embedded in paraffin, and tissues were stained with H and E.

All animal experiments were conducted in accordance with the principles of the NIH Guide for the Care and Use of Laboratory Animals and approved protocols of the Institute of Materia Medica, CAMS & PUMC, Beijing, China. Female BALB/c-nu nude mice (16–18 g) were bought from Vital River Laboratory Animal Technology (Beijing, China) and housed at the Institute of Materia Medica animal barrier facility. Xenografting was done by subcutaneous implantation of U87 cells (1 × 10⁷/0.1 mL) into the right flanks of the mice. After 12 days, when the tumor volume had reached about 100 mm³, the mice were randomly divided into three groups (n = 6) and treated with SW33 once a day for three weeks (30 and 60 mg/kg, i.g.). Body weight and tumor volume were recorded every two days. The tumor volume (V, mm³) was calculated by Eq. (2): $V=0.5\times a\times b^2$ where a represents length and b represents width, in mm. At the end of the experiment, the mice were euthanized and blood was collected by capillary from the medial canthus of the eye. Complete blood count (CBC) was measured using a hematology analyzer (MEK-7222K, Nihon Kohden). The weight of the tumor tissues and the heart, liver, spleen, kidney, lung and brain were measured.