User:Z3333429

From CellBiology

z3333429

Attendance

Lab 1: Z3333429 (talk) 16:34, 12 March 2015 (EST)

Lab 2: Z3333429 (talk) 16:05, 19 March 2015 (EST)

Lab 3: Z3333429 (talk) 16:04, 26 March 2015 (EST)

Lab 4: Z3333429 (talk) 16:15, 2 April 2015 (EST)

Lab 5: Z3333429 (talk) 16:04, 16 April 2015 (EST)

Lab 6: ABSENT

Lab 7: Z3333429 (talk) 16:20, 30 April 2015 (EST)

Lab 8: Z3333429 (talk) 15:58, 7 May 2015 (EST)

Lab 9: Z3333429 (talk) 16:26, 14 May 2015 (EST)

Lab 10: Z3333429 (talk) 16:25, 21 May 2015 (EST)

Lab 11:

Lab 12:

Lab Assessment 1

PubMed

http://php.med.unsw.edu.au/cellbiology/index.php?title=Cells_Eukaryotes_and_Prokaryotes

Cells Eukaryotes and Prokaryotes

PMID 25513760

<pubmed>25513760</pubmed> SEMbacteria.jpg

Scanning Electron Micrographs of S. aureus (fig. A and B) and B. subtilis (fig. C and D) that were treated with a cationic defensin (SibaDef) so as to reveal morphological changes.[1]


Lab Assessment 2

Isotropic 3D Super-resolution Imaging with a Self-bending Point Spread Function[2]

SUMMARY:

A self-bending point spread function (SB-PSF) was adapted using existing Airy beam technology with the aim of creating super-resolution fluorescence imaging in three dimensions (3D). This method produced a side-lobe-free SB-PSF, decreasing diffraction and lateral bending and allowing for super-resolution imaging with accurate 3D localisation of molecules over an increased image depth and higher localisation precision. This means that the SB-PSF method can image thicker samples with a much higher resolution then other current models.


METHOD:

  • The optical set up included taking measurements with custom inverted microscope that was calibrated for internal reflection and oblique incidence excitation.
  • Filtered fluorescence light was then passed through activated dye samples.
  • The images were aligned between two channels and imposed with markers. They were then displaced in 100nm increments over a distance of roughly 3 microns.
  • Images of ten regions were superimposed to create an image with a concentration of markers in 3D.
  • The fiducial markers were then illuminated with a laser and were recorded as STORM movies.
  • Single molecule and STORM movies were separated and analysed and any images that did not meet the set criteria were rejected.
  • Results were conducted on the basis of localisation precision, image depth and error rates.


RESULTS and CONCLUSION:

The SB-PSF model can create super-resolution fluorescence images of depths 1.5-10 times larger than other current methods and 2-3x higher localisation precision. The main advantages of this method are the ability to scan thicker samples at higher resolution whilst preventing the effects of photo bleaching.

One downside of this new adaption is that the larger scanning area results in a small decline in imaging velocity due to the reduction in the number of fluorophores per frame. Another drawback is the lower density of activated fluorophores revealed by larger depth scans. In order to optimise the resolution, combining SB-PSF with ultra-bright photoactive fluorophores could help to distinguish a greater amount of photons and create images with incredibly high resolutions in nanoscale.

Lab 3

Paraformaldehyde

Commercial MSDS: Paraformaldehyde MSDS - Sigma-Aldrich

PubMed

Search term: Collagen Type II

<pubmed limit=5>Collagen type II</pubmed>

BioMed Central

http://www.biomedcentral.com/search/results?terms=collagen+type+II

Lab 3 Assessment

Article 1

The effect of estrogen on the expression of cartilage-specific genes in the chondrogenesis process of adipose-derived stem cells.[3]

SUMMARY: The aim of this study was to determine the effect of oestrogen on genes pertaining to chondrogenesis. Adipose-derived stem cells (ADSCs) were differentiated into cartilage and then treated with oestrogen in order to determine the genetic markers associated with the expression of type II collagen.

METHOD:

  • Cell culture: Human adipose-derived stem cells were sourced from patients aged 25-55 years using enzymatic digestion of subcutaneous tissues.
    • The cells were then cultured in a modified medium that was changed two times a week allowing for differentiation into cartilaginous tissues.
  • Pellet culture: Pellets of chondrogenic APSCs were formed using a centrifuge and then suspended in a treated chondrogenic medium.
    • The pellets were then incubated for two weeks and the control group was left untreated whilst the experimental group was treated with oestrogen E2.
    • At this point in the procedure the medium was changed every three days.
  • Reverse transcription polymerase chain reaction (RT-PCR): Gene expression of cartilage-specific markers was determined using the RT-PCR method.
    • RNX-plus kits were used to extract the cellular RNA and any unwanted genomic DNA was removed through the use of DNase.
    • Concentrations of RNA were assessed using a spectrophotometer and cDNA was synthesised from the identified RNA and used for PCR.

RESULTS and CONCLUSION: Type II collagen was found in the control group but no type II collagen was observed in the experimental group. Aggrecan was detected in both groups with a significant decrease in aggrecan expression in the experimental group.

The study demonstrated that oestrogen has an inhibitory effect on the expression of type II collagen and also leads to a significant reduction in aggrecan gene expression. This means that oestrogen is not suitable for use in the chondrogenesis of type II cartilage from ADSCs.

Article 2

Type 1 regulatory T cells specific for collagen type II as an efficient cell-based therapy in arthritis.[3]

SUMMARY: The aim of this study was to assess the potential of collagen type II regulatory T cells (Col-Treg) for treatment of rheumatoid arthritis (RA).

METHOD:

  • Ethical guidelines were followed and approved of by appropriate ethic committees.
  • BALB/c and DBA/1 mice were obtained and separated into transgenic groups of rearranged T-cell receptors (TCRs) and type II collagen specific T-cell hybrids.
  • Col-Treg clones were generated from Col II–specific transgenic mice.
  • Ova-Treg clones were generated were also generated.
    • IL-10 was added to both groups on day 2 and the cells were then cloned.
  • Cytokines IL-4 and interferon γ (IFN- γ) were counted using an enzyme-linked immunosorbent assay (ELISA) after two days of stimulation.
    • Cytokine secretions were stained for using fluorescent dye and then analysed using fluorescence-activated cell sorting.
    • The immunosuppressive function of Col-Treg clones was then assessed.
  • Arthritogenic antibodies specific for collagen type II were injected intraperitoneally into 9 week old DBA/1 mice.
    • Col-Treg cells were delivered via intravenous injection into the mice hours later.
    • Beginning from 3 days post initial injection, the disease severity was scored using a scale:

0 = normal 1 = weak swelling 2 = significant swelling associated with redness 3 = intermediate swelling associated, or not, with redness 4 = maximal swelling and/or redness in all inflamed digits

  • The number of inflamed digits were taken into account and scored appropriately (0 = no inflamed digits, 0.5 = zero to five inflamed digits, 1 = six to ten inflamed digits, 1.5 = one to fifteen inflamed digits and 2 = 16 or more inflamed digits).
    • The mice were also weighed to determine any lose in body weight.
  • 9-12 week old DBA/1 mice were immunised at the base of the tail with bovine type II collagen.
    • 21 days after the arthritis tests, a booster shot was given.
    • Col-Treg cells were injected intravenously into the mice on day 20, 22 or 28 after arthritis induction.
    • The thickness of each hind paw was measured consistently after day 21 and the severity of arthritis was graded.
  • Samples were collected and stained.
    • Leukocyte infiltration and erosion were scored according to appropriate scales.
  • Immunoglobins were measured.
  • Detection of Col-Treg cells was carried out using PCR.
  • 9 week old BALB/c mice were injected with ova-specific CD4 cells.
    • This group was then immunised the following day with IFA.
    • On day 5 one paw was injected with ovalbumin/phosphate-buffered and PBS was injected into another.
    • Specific T regulatory cells were injected intravenously and the mice were killed after 2 days. Samples were then collected and stained.

RESULTS and CONCLUSION:

The results indicate that introduction of Col-Treg cells reduces the incidence and clinical symptoms of arthritis in both preventive and curative settings. There was a significant impact on collagen type II antibodies and there was a noticeable decrease in antigen-specific effecter T cells. These results indicate that collagen type II T regulatory cells could be an effective treatment from patients suffering from RA.

Article 3

Remission of Collagen-Induced Arthritis through Combination Therapy of Microfracture and Transplantation of Thermogel-Encapsulated Bone Marrow Mesenchymal Stem Cells.[3]

SUMMARY: This study provided a new therapeutic strategy for autoimmune inflammatory diseases such as Rheumatoid Arthritis particularly with the effect on collage type II.

METHOD:

  • 36 male Sprague-Dawley (SD) rats were used for the experiment and ethical guidelines were followed to keep suffering minimal.
  • The SD rats were divided into for groups: CON, BLA, GEL and BMC. *All groups were injected subcutaneously with collagen type II emulsion. *Booster shots were given at d=21 with half the dose.
  • All groups received operations one week after booster shots.
  • In the BLA, GEL, and BMC groups a hole was drilled into the tibial plateau of the left knee and then the wound was closed.
  • The CON group received a sham operation with no drilling performed. *The rats were then allowed to move freely after their respective operations and were monitored.
  • Bone marrow was harvested from the tibia and femur of a 3 week old SD rat and the mononuclear cells were isolated. The bone marrow mesenchymal stem cells (BMMSCs) were prepared for use.
  • The groups all received different treatments 3 days post surgery.
    • BMC: PLGA-b-PEG-b-PLGA (BMMSCs); GEL: thermogel ; BLA (Blank group): phosphate-buffered saline (PBS); CON (control group): PBS
  • The rats were then scored in terms of arthritis onset according to level of erythema, swelling or joint rigidity and oedema. An average of the limbs was calculated to determine scores.
  • The rats were killed and both distal femurs were examined and photographed for macroscopic evaluation.
  • The distal femurs were then fixed and stained in order to perform microscopic of the knee cartilage and surrounding synovium.

RESULTS and CONCLUSION: The results indicated that the majority of SD rats developed some form of irreversible bone or cartilage degradation with the exception of the BMC group. The BMC group displayed scores significantly lower than the non-treatment groups but were still possessed higher degrees of disease than normal rats. This means that BMMSC therapy can reverse synovial hyperplasia to an extent but cannot offer a full recovery.

Article 4

TGF-β1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells. [3]

SUMMARY: The purpose of this study was to test the effectiveness of a biofunctional hydrogel consisting of collagen type II nanofibers and transforming growth factor β1 (TGF-β1) in the regeneration of cartilage.

METHOD:

  • Photocrosslinkable hydrogels were prepared.
  • TGF-β1 was added to the hydrogels at a concentration of 10 μg/mL.
  • Human synovium-derived mesenchymal stem cells (hSMSCs) were suspended in hydrogels and then cultured in chondrogenic medium.
  • Growth of hSMSCs was observed with a light microscope.
  • Cell viability was observed by staining the samples after washing with PBS and viewing them via fluorescent microscopy. Viability was determined by the ratio of live cells to total cells.

RESULTS and CONCLUSION: The results indicate that collagen type II impregnation and TGF-β1 delivery significantly promoted chondrogenesis. This hydrogel system could be an effective treatment for cartilage defects and the results support the hypothesis that collagen type II impregnation in conjunction with TGF-β1, promote chondrogenesis in hSMSCs.

Image

Articular cartilage of rat knees treated with BMMSCs.jpg

Macroscopic Comparison of Rat Knee Articular Cartilages After Treatment with BMMSCs.[4]

Lab 5 Assessment

Analysis B35 neuro-epithelial cells Group 2 Graph.jpeg


This graph shows the data collected after the actin cytoskeleton of B35 neuro-epithelial cells was manipulated by Tropomyosin 4 (Tm4). The cells were categorised into five phenotypes: fan, broken fan, stumped, pronged, stringed, and pygnotic. Increased numbers of 'pronged' and 'stringed' phenotypes after Tm4 expression shows that Tm4 is involved in promoting the formation of neutrites. Tm4 overexpression stabilises actin filaments and thus facilitates the formation of neutrites.

Lab 6 Assessment

Collagen II Antibody MA5-37493 MA5-37493

Species: Mouse

Working concentration: Western Blot: 1-2 µg/ml

Imunofluorescence: Assay Dependent

Immunohistochemistry: 1-2 µg/ml

Flow Cytometry: Assay Dependent

Secondary antibody: Biotinylated secondary antibody to rabbit IgG[5]

Paper that used Collagen Antibody MA5-37493: Articular cartilage increases transition zone regeneration in bone-tendon junction healing.[5]

Lab 9

Mouse Cell Line: L Cells (ATCC® CRL-2648™)

Culture Media: Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.

Components of Dulbecco's Modified Eagle's Medium

Human Cell Line: 20B8 (ATCC® CRL-12582™)

Culture Media: RPMI-1640 Medium, Catalog No. 30-2001. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.

Components of RPMI-1640 Medium


Penicillin-Streptomycin The antibiotics penicillin and streptomycin are used to prevent bacterial contamination of cell cultures due to their effective combined action against gram-positive and gram-negative bacteria. Penicillin was originally purified from the fungi Penicillium and acts by interfering directly with the turnover of the bacterial cell wall and indirectly by triggering the release of enzymes that further alter the cell wall. Streptomycin was originally purified from Streptomyces griseus. It acts by binding to the 30S subunit of the bacterial ribosome, leading to inhibition of protein synthesis and death in susceptible bacteria.

References

  1. <pubmed>25649358</pubmed>
  2. <pubmed>25383090</pubmed>
  3. 3.0 3.1 3.2 3.3 <pubmed> 25789269</pubmed> Cite error: Invalid <ref> tag; name "”PMID" defined multiple times with different content Cite error: Invalid <ref> tag; name "”PMID" defined multiple times with different content Cite error: Invalid <ref> tag; name "”PMID" defined multiple times with different content
  4. <pubmed>25774788</pubmed>
  5. 5.0 5.1 <pubmed>18987921 </pubmed>