A total of 20 amyloid precursor protein (APP)/presenilin-1 (PS1) double transgenic mice (Beijing Biocytogen Co., Ltd., Beijing, China) were randomly divided into two groups: the AD control group (n=10) and the stem cell treatment group (n=10). Ten C57 black 6 bred in the Jackson Laboratory (C57BL/6J) non-transgenic mice were included in the normal control group. The mice were kept in cages with controlled temperature and light cycles (24°C and 12/12 light cycles) and had free access to food and water. The humidity was 60±10%. There were no statistically significant differences in age, sex and weight among the three groups of mice (P>0.05), and the data were comparable (Table I). The study was approved by the Ethics Committee of the Second Hospital of Shandong University (Jinan, China).

Cell culture Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and trypsin powder were purchased from Gibco (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNAiso Plus, PrimeScript^® Real-Time (RT) Reagent kit with genomic deoxyribonucleic acid (gDNA) Eraser (Perfect Real-time) and SYBR^® Premix Ex Taq™ II [Tli ribonuclease H (RNase H) Plus] were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Zhongshan Golden Bridge Biotechnology Co., Ltd.; OriGene Technologies, Beijing, China).

Operations were conducted under aseptic environment. A 200-mesh sieve was used for cell screening. The parameters of cell and tissue centrifugation for the separation process were 800 × g/min at 4°C for 3 min. Phosphate-buffered saline (PBS) was used for thorough washing, as the clearer and cleaner the solution was the better the results would be. The washing was conducted each time for approximately ≥2 times. In addition, the number of living cells was determined using trypan blue staining, and counted using a cell counter. Cell culture reagents, consumable items and environmental conditions (temperature and humidity) were strictly adjusted in accordance with the characteristics of the cell growth. After the cell growth area reached ~80% of the total area of the flat bottom, the passage could be performed, and the passage ratio would be determined according to the amount of cells required for the next experiment.

Flow cytometry detection (10): i) the test objects were the fourth-generation BMSCs. ii) The centrifugation parameters for the preparation of the flow cytometry samples were 800 × g/min at 4°C for 3 min. iii) The cells were mixed and suspended in PBS, and the number of cells was 1–2×10⁶. ⅳ) Surface antibody (4 µl) was added in each tube and incubated at 25°C for 30 min avoiding light. v) PBS (500 µl) was used to wash the cells. Then the cells were collected and centrifuged at 800 × g for 5 min. The supernatant was discarded. vi) PBS (500 µl) was added for resuspension. Then the cells were added to the BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). ⅶ) The cell flow rate was moderate, and the cell population conforming to the characteristics was selected according to forward scatters (FSCs) and side scatters (SSCs). ⅷ) The voltage was adjusted according to the graphics to make the graphics easy to observe and for better display results.

BMSC suspension was prepared. Stem cells for intravenous injection were digested from the adherent state to the suspended state, and washed with normal saline, followed by centrifugation at 800 × g/min at 4°C for 3 min twice and filtering by a cell sieve filter once. Then the filtered stem cell suspension was immediately placed on ice and transferred to the intravenous injection site. The mice aged 7 weeks were fixed in a rat fixator, and 1 ml suspension containing 2×10⁶ stem cells was injected using a 2.5 ml syringe each time. The normal control group and the AD control group were injected with the same volume of normal saline once a day.

The experiment began on the 22nd day after gavage treatment and was divided into two stages. In the first stage, mice received 3-day continuous training twice a day, and the experimental time was not recorded. In the second phase, mice underwent the fixed-position cruise for 5 consecutive days, once daily: the mice were placed facing the wall of the pool and put in pools from different quadrants to record the time they searched for the underwater surface hidden in the third quadrant (escape latency). If the mice could not find the platform within 90 sec, they were guided to the platform for a rest for 30 sec, and the latency was recorded as 90 sec. At the end of fixed-position cruise, the space exploration experiment was conducted for 1 day: the platform was withdrawn, the time occupied by the mice in the third quadrant within the 90 sec and the frequency of crossing the original platform were recorded. Morris water maze image automatic monitoring and processing system was applied to collect and process data.

ELISA kits were used to detect the contents of interleukin (IL)-1, IL-2, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in plasma as well as amyloid β (Aβ)1–42 content in brain tissues. The relative expression levels of β-site APP cleaving enzyme 1 (BACE1) and α-2-macroglobulin (A2M) genes were detected by fluorescence quantitative polymerase chain reaction (qPCR), and the primer sequences are shown in Table II. mRNA levels were determined by Bio-Rad CFX96 fluorescence qPCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The thermocycling conditions were as follows: 5°C for 5 min; 94°C for 10 sec, 50°C for 30 sec, 40 cycles; 72°C for another 10 min. The image was analyzed with GraphPad Prism statistical software (GraphPad Software, Inc., La Jolla, CA, USA).

All data were processed using Statistical Product and Service Solutions (SPSS) 16.0 software (Cabit Information Technology Co., Ltd., Shanghai, China). Measurement data were expressed as mean ± SD, and the comparison of mean was conducted using t-test and one-way analysis of variance. The post hoc test used was LSD test. P<0.05 was considered to indicate a statistically significant difference.

BMSCs were successfully isolated. They were spindle-shaped and spirally arranged, and grew adhering to the culture dishes (Fig. 1). The obtained BMSCs were identified using a flow cytometer, which revealed that cluster of differentiation (CD)29, CD44 and CD90 were positive, whereas CD45 and human leukocyte antigen antigen-D-related (HLA-DR) were negative (Fig. 2).

From day 2, the escape latency in the water maze in the stem cell treatment group was shortened compared with that in the AD control group (P<0.05). From day 3, there was no significant difference in the escape latency between the stem cell treatment group and the normal control group (P>0.05) (Fig. 3). The space exploration experiment showed that the time of crossing the ring for the first time in the stem cell treatment group was shorter than that in the AD control group (P<0.05), but the frequency was higher than that in the AD control group (P<0.05) (Table III).

Aβ1–42 content in the AD control group was higher than that in the stem cell treatment group and the normal control group (P<0.05). The expression level of BACE1 in the stem cell treatment group was lower than that in the AD control group (P<0.05), while the expression level of A2M gene was increased in the stem cell treatment group compared to that in the AD control group (P<0.05) (Fig. 4).

At 14 days after treatment, the contents of IL-1, IL-2, TNF-α and IFN-γ in blood in the stem cell treatment group were lower than those in the AD control group (P<0.05) (Table IV).