| Approximately 70%-85% of individuals experiences low back pain (LBP) during their lifetime. LBP and subsequent disabilities are common. Intervertebral disc degeneration (IVDD) is considered a major source of LBP. IVD tissue is composed of three highly specialized parts: Cartilage End-plate(CEP),Anulus Fibrosus(AF) and Nucleus Pulposus(NP). NP is the first changed structure during IVDD. In our body NP is the largest avascular tissue, and its cell number is limited, so it is difficult to restore function by itself. To date, most currently available surgical options for IVDD can not recover the normal function of IVD.Along with the establishing of IVD NP and AF cell culture technique in vitro, and the development of the study of IVD cells from different species and also the study of the cartilage tissue engineering, it has brought us a promising future for the function regeneration of NP. The NP tissue engineering has been a direction of biological treatment for IVDD. In the study of tissue engineering, the culture of the seed cells is essential. However, because of the slower growth and complex culture technique of NP, it is very difficult to get large numbers of NP cells. In addition, there is no consensus about the NP cell phenotype. All of above limit the development of NP tissue engineering. We want to modify NP cells by gene engineering to solve the cell source problem. So we try to use hTERT (human Telomerase Reverse Transcriptase) gene mediated by lentivirus gene vector to immortalize NP cells and to obtain enough NP cells that have stable character.During culturing of NP cells, we found that there were many spindle-shaped MSC (Mesenchymal Stem Cell)-like cells. They were proliferating. Some samples even can passage to the 10th passage and remaining well growth. So we characterized these spindle-shaped cells according to the MSC characterization standard and to verify whether we can isolate and identify the NP-MSC (Nucleus Pulposus derived Mesenchymal stem cells). Above this we compared further the differences of NP cell and NP-MSC and of NP-MSC and BMSC (Bone Marrow Mesenchymal Stem Cells) from the same subject. All the experiments can be divided into 4 sections:Section 1: Packaging hTERT Lentivirus and its Titer MeasuringIn this section, we use plasmid pMD18-T-hTERT as template to amplify the target gene hTERT by PCR method. Then the lentivirus eukaryotic expression vector pGC-FU-EGFP-3FLAG was purified by large amount. After enzyme digestion linearization of endonuclease AgeI and NheI and purification, under the in-fusion exchange enzyme condition, the target gene hTERT was exchanged and ligated into the linearized vector. It was transformed into E.coli DH5αthrough CaCl2 protocol. The new colonies were screened and characterized by colony PCR. Enlarge the positive transformer culture and then sequence them. The sequencing results indicate that the target gene in the positive colony is the same as the original completely. It was proved that we get the hTERT lentivirus eukaryotic expression vector pGC-FU-TERT to use as virus packaging. Thaw and passage the 293T cell (using logarithmic phase). Transfect the 3 plasmids (pGC-FU- hTERT,pHelper1.0,pHelper2.0) into 293T cell by Lipofectamine2000 together. After 48h, the cultured supernatant was collected. That is virus extraction. It was condensed though high speed centrifugation settings. The titer of the condensed virus was 2E+8 TU/mL detected by Real-time fluorescence qualification PCR. Western Blot experiment was carried out to testify the expre- ssion of target gene hTERT in 293T. The results proved to get the higher titer of Lenti-hTERT. This is prepared for infecting human NP cells.Section 2: Infecting Human Nucleus Pulposus cells with Lenti-hTERT and Identifying the Infected CellsIn the second section, firstly, the 2 NP samples of idiopathic scoliosis (IS) we collected were 10y and 14y. The normal human NP cells was isolated and cultured via enzyme digestion. We observed the morphology of the NP cells, and detected the expression of collagen typeⅡby immunohistochemistry ABC protocol. Also CD24 expression of NP cells was measured by Flow Cytometry. The results were that the NP cells were spindle-shaped, and the collagen typeⅡstaining and CD24 expression were all positive. Indicate that we successfully isolated the normal human NP cells in vitro.Secondly, preliminary experiment had been done using Lenti-GFP. We infected this virus which titer is 2E+9 TU/mL to cultured human NP cells according to different MOI (100, 50, 10, 1, respectively). There were two groups: with polybrene and without polybrene. After infection, through the fluorescent microscopy and Flow Cytometry, we got the infection condition and parameter: the infective effectiveness was best when MOI is 100, and need not polybrene. The infected cells were passged to the 5th, the positive rate of GFP can remain above 60%. That is to say GFP gene mediated by lentivirus can express in NP cells stably and long term.Thirdly, after infection with the condensed Lenti-hTERT act on the above infection condition, the human NP cells were detected the telomerase expression via Western Blot. Apart from the positive results, the telomerase activity was also higher than before infection. Compared with the non-infected group, the passage property improved obviously. They could be passged to more than 10 passages, whereas the non-infected NP cells will appear the"dedifferentiation". At the same time, the infected NP cells can remain the same character as the non-infected ones: the cell morphology, the immunohistochemistry staining and also the CD24 expression. Because the GFP gene was not constructed into the Lenti-hTERT, we screened the positive cells through the continuous passaging method. After several passages, the cells that can express hTERT would become more. The cells we collected to detected telomerase activity were cultured above 6 passages.These results verified that we had obtained the immortalized human NP cells. The infected cells were also proved to have no oncogenicity with nude mice. It could provide some experimental proof for the human NP cells modified with lentivirus vector used as seed cellsSection 3: Comparision of Intervertebra Disc Nucleus Pulposus Cells with Nucleus Pulposus-derived Stem Cells (NP-MSC)Nucleus Pulposus-Mesenchymal Stem Cells (NP-MSC) were isolated and characterized from 7 patients (5 of IS, 2 of degenerated disc) undergoing various spinal operations. We collected the 5th spindle-shaped cells, compared with the polygonal cells from the first two passages. The studying cells were all from the larger and healthier cell colonies. The morphologies, mRNA levels of several extracellular matrix markers (Collagen typeⅡ, aggrecan and SOX9) for NP, immunophenotypes (CD105, CD29, CD44, CD34, CD14, CD45, HLA-DR and CD24), and differentiation abilities (osteogenic, adipogenic and chondrogenic ability) of the isolated cells were examined.Results: The spindle-shaped cells isolated from NP tissue were proliferate, appeared similar morphology with the MSC from BM or other sources. After several passages, they can remain the original appearance, whereas the polygonal cells showed"dedifferentiation", senescence and low proliferate. The immunophenotype of the spindle-shaped cells were positive for CD105, CD29 and CD44, negative for CD34, CD14, CD45, HLA-DR and CD24. Opposite to the spindle-shaped cells, the polygonal cells were partly positive for CD24, CD44, CD29, negative for the other antigens. In the differentiation experiments, the spindle-shaped cells had the osteogenic, adipogenic and chondrogenic differentiation ability except one 37Y case in adipogenic ability. But the polygonal cells showed to die or to be dying in differentiation medium. They had not the differentiation ability.Among our samples, those from 10Y and 14Y can be transferred over 10 passages, while others only to 5-6 passages and became aged. The polygonal cells of first 2 passages, when transferred to after 4 passages, became less and less, whereas the spindle-shaped cells got more and more. So we believed that maybe the polygonal cells are NP cells, whereas the spindle cells that have the multi-linage differentiation ability and the cell surface phenotype of MSC should be MSCs derived from NP.Section 4: Comparisons of Nucleus Pulposus-derived Stem Cells (NP-MSCs) with Bone Marrow Mesenchymal Stem Cells (BMSCs) from the Same SubjectIn this section, 3 cases were both isolated their BMSCs and NP-MSCs from the same subject. Compared with their morphology and passage cycle (replate about 2×105 cells into the 25cm2 culture bottle, when they are 90% confluent, it can be taken as one passage, continue the same operation after tripsin treatment), examined the cell phenotype by Flow Cytometry, and the osteogenic, adipogenic and chondrogenic differentiation ability.Results: morphology of two types MSC from the same subject was similar extremely. They were both spindle-shaped and adherence to plastic culture bottle. Their passage cycle had some differences: it took 6-9 days every passage for NP-MSC and 4-6 days for BMSC. The older samples would take more time even over 10 days. As to the phenotype, they were both positive for CD105, CD29 and CD44. But the differences were that the mean fluorescence intensity (MFI) of the 3 positive markers of NP-MSCs were all weak than those of BMSCs. They were both negative for other hematopoietic markers.There were no obvious differences between the NP–MSCs and BMSCs in the osteogenic induction experiments. Both were observed the calcium nodus after inducing 14-18 days. In the chondrogenic differentiation, the round cell pellets forming time were different and the cell pellets were all from same number of cells. It took NP–MSCs about 3-6 days, whereas BMSCs above 12 days. There may be some differences between the two cell types in the chondrogenic differentiation. Adipogenic differentiation: BMSCs formed oil droplet faster and more obvious. We could observe the droplet from microscopy without staining about 8 days'induction. NP-MSCs could not be observed even after 12 days'induction. After 16 days and staining, we could find the smaller oil droplet. Those cells appeared disintegrating (Figure 4-4). The viability of adipogenic these cells were far lower than that of BMSCs.Conclusions:1. Lentivirus expression vector pGC-FU-hTERT was constructed successfully. High titer of lentivirus Lenti-hTERT was condensed after infecting target cells 293T.2. NP cells were infected successfully by Lenti-hTERT and could express telomerase. The modified NP cells could passage many times stably and maintain the basic character of NP cells. The immortalized NP cells could be used as seed cells for NP tissue engineering.3. MSC-like cells were isolated successfully from NP tissue and were initially characterized that they had the property of MSC. 4. MSC-like cells and NP cells isolated from the same NP tissue had obvious differences in shape, cell surface phenotypes and extracellular matrix secreting. It could demonstrate that there are both NP cells and NP-MSCs in NP tissue.5. NP–MSCs and BMSCs from the same subject may have some differences in chondrogenic and adipogenic differentiation. NP–MSCs may have better application values in chondro- genic inducing since it can be used as a new type of seed cells in tissue engineering. |
