The Study Of IGF-1 Promoting The Proliferation And Differentiation Of Human Dental Pulp Cells Via JAK-STAT Signaling Pathway In Vitro

Objective: To investigate the effects of JAK-STAT signaling pathway on the proliferation and differentiation of human dental pulp cells(HDPCs) which were induced by insulin-like growth factor 1(IGF-1).Methods: Isolation, culture and identification of human dental pulp cells. We collected healthy teeth which were extracted freshly for orthodontic reasons from young adults. The tissue block and enzymatic digestion method was used to isolate and culture primary HDPCs. Cells were detached with 0.25% trypsin when they reached 80% confluence and subcultured to fisrt passage. Only cells from third or forth passage were used in the further experiment. Then we used immunohistochemical staining of vimentin and keratin to determine origin of cells.The effect of IGF-1 on proliferation of HDPCs.Cells were seeded at a density of 3×103cells per well in a 96-well plate and cultured for 24 h. Then these cells were serum-starved for 24 h. IGF-1 at 5, 10, 25, 50 and 100ng/ml was added separately to DMEM containing 2% FBS in the experiment groups. The culture medium was refreshed every 3 days. The cells were incubated for 1,3, 5 and 7 days,and then treated with 20μl of MTT for 4 hour. MTT solution was removed and replaced with 150 μL of DMSO. The absorbance at 490 nm was read with an automatic enzyme-linked immunosorbent assay reader.The effects of JAK-STAT signaling pathway on the proliferation of HDPCs enhanced by IGF-1.Cells were seeded at a density of 3×103cells per well in a 96-well plate and cultured for 24 h. Then change the medium with five medium containing 2% FBS(control, blank control, medium containing 100ng/ml IGF-1 with or without AG490, and medium containing AG490 alone). The cells were incubated for 1,3, 5 and 7 days,and then use the MTT assay to explore proliferation of cells.Alkaline phosphatase staining and alizarin red staining were used to determine the effect of IGF-1 on the osteoblast differentiation of HDPCs.The third passage HDPCs were seeded at a density of 2×105cells per well in a 6-well plate. After growing to 80% confluence, cells were incubated in four kinds of medium(including mineralizing medium, mineralizing medium with 100ng/ml IGF-1 or AG490, and mineralizing medium containing both 100ng/ml IGF-1 and AG490). After incubated for 7,14 and 21 days, the cells were fixed with 4% paraformaldehyde for 30 min. The degree of extracellular matrix calcification was estimated using an Alizarinred S and Alkaline phosphatase staining. The amount of calcium deposition was quantified by destaining with 10% cetylpyridinium chloride monohydrate. The absorbance was measured at 562 nm.Real-time reverse transcription-polymerase chain reaction(Real-time RT-PCR) was used to detect the m RNA expression of osteoblast and odontoblast related genes.After 14 days of culture with four kinds of mineralizing medium above, cells were harvested using 1 ml TRIzol reagent. Total RNA was extracted according to the manufacturer’s protocol. The first-strand c DNA was synthesized using a M-MLV first-strand c DNA synthesis kit. Then we used RT-PCR to detect the m RNA expression of ALP, OCN, OPN and DSPP.Western blot assays and immunofluorescent staining were used to detect expression of phospho-JAK2.After treatment with four kinds of mineralizing medium above for 4 hours, cells were rinsed with cold PBS and lysed in RIPA buffer containing phosphatase inhibitors. Firstly, all sample total protein extracted were run on SDS-Tris-HCl Ready Gels, and then transferred onto PVDF membrane. Secondly, membranes were blocked with milk blocking solution, and subsequently incubated with primary antibodies(phosphor-JAK2, 1:1000; GAPDH, 1:1000). Thirdly, membranes were incubated with appropriate secondary antibodies(1:20,000). Finally, membranes were scanned by double color infrared laser scanner. The results were integrated using gel analyst software(Image J).After treatment with four kinds of mineralizing medium above for 4 hours, cells were fixed with 4% paraformaldehyde for 30 min, and then blocked for 30 min with 10% FBS. Cells were incubated with primary antibodies(p-JAK2, 1:100) and secondary antibodies(1:1000). Finally, nuclei were stained with DAPI. The cells were examined with a Zeiss fluorescence microscope.Statistics were calculated using SPSS 13.0. Results are expressed as the mean±standard deviation. Differences between groups were tested for statistical significances using One-Way ANOVA. Comparisons between means were assessed using S-N-K.A P value of < 0.05 was considered significant.Results:1 Human dental pulp cells were isolated and cultured in vitro successfully. The cells were identified mesenchymal origin with the result that cells were stained positive for vimentin and negative for cytokeratin.2 The 5–100ng/ml of IGF-1 promoted proliferation of HDPCs. When cells were cultured 5 days or 7 days, IGF-1 at 25–100ng/ml enhanced proliferation of HDPCs compared with the control groups(P<0.05). IGF-1 at 100ng/ml presented the highest activity of proliferation among all groups(P< 0.01). The group containing 100ng/ml of IGF-1 and AG490, inhibited significantly proliferation of HDPCs as compared with the group containing IGF-1(P<0.01).3 Treated group with 100ng/ml IGF-1 presented a higher positive cells ratio of ARS and ALP staining at day 7, 14 and 21 than Control/M(P<0.05). However, the group with 100ng/ml IGF-1 and AG490 presented a lower positive cells ratio of ARS and ALP staining than group with 100ng/ml IGF-1(P<0.05).4 The ALP,OCN,OPN and DSPP m RNA levels were dramatically increased in cells exposed to osteogenic induction medium with IGF-1 for 14 days(P< 0.05). The expression of these genes were significantly down regulated in group containing IGF-1 and AG490 which were compare to group containing IGF-1. These results were consistent with Alizarinred S and Alkaline phosphatase staining.5 After treatment with four kinds of mineralizing medium above for 4 hours, cells were detected by Western blot assays and immunofluorescent staining. The results showed that the levels of p-JAK2 in IGF-1 treating cells were upregulated as compared with Control/M. In the contrary, both IGF-1 and AG490 treated HDPCs exhibited lower protein expression levels of p-JAK2 as compared with group of IGF-1.Conclusions:1 The HDPCs were successfully cultivated by the method of tissue explant-collagenase digestion. They were indentified as mesenchymal origin by immunohistochemistry staining. The third and forth passages HDPCs which had higher cell proliferation and well cell morphology meet the requirement of the experiment.2 IGF-1 of 25–100ng/ml can stimulate the proliferation of HDPCs in a concentration-dependent manner. Furthermore, 100ng/ml IGF-1 was the optimal concentration and was used for subsequent experiments.3 IGF-1 treated HDPCs exhibited higher protein expression levels of p-JAK2. IGF-1 induced proliferation and osteoblast differentiation of HDPCs through the activation of JAK-STAT signaling pathway. As the same time, IGF-1 can trigger the m RNA expression of osteogenic differentiation(ALP、OCN、OPN) and odontoblast differentiation(DSPP)in HDPCs.